Competence remodels the pneumococcal cell wall exposing key surface virulence factors that mediate increased host adherence

Competence development in the human pathogen Streptococcus pneumoniae controls several features such as genetic transformation, biofilm formation, and virulence. Competent bacteria produce so-called “fratricins” such as CbpD that kill noncompetent siblings by cleaving peptidoglycan (PGN). CbpD is a choline-binding protein (CBP) that binds to phosphorylcholine residues found on wall and lipoteichoic acids (WTA and LTA) that together with PGN are major constituents of the pneumococcal cell wall. Competent pneumococci are protected against fratricide by producing the immunity protein ComM. How competence and fratricide contribute to virulence is unknown. Here, using a genome-wide CRISPRi-seq screen, we show that genes involved in teichoic acid (TA) biosynthesis are essential during competence. We demonstrate that LytR is the major enzyme mediating the final step in WTA formation, and that, together with ComM, is essential for immunity against CbpD. Importantly, we show that key virulence factors PspA and PspC become more surface-exposed at midcell during competence, in a CbpD-dependent manner. Together, our work supports a model in which activation of competence is crucial for host adherence by increased surface exposure of its various CBPs.


3) On occasion it is not clear what statements are based on literature vs representing a hypothesis
We do not state in the paper that CBPs attach preferentially to either WTA or LTA. Only that the differential expression of LytR, ComM and TacL leads to differing ratios of WTA/LTA amounts in the cell wall.
c: Please clarify if there is literature evidence that LytR attaches TA to cell wall in addition to the results that are compatible with but do not directly show it in this paper: We have now cited a study that showed that deletion of lytR led to decreased retention of TAs to the pneumococcal surface (Ye et al. 2018).
Reviewer 2: 1) 2. Reverse "surface exposing": We have rephrased the title to help clarify title meaning to: Competence remodels the pneumococcal cell wall providing resistance to fratricide and exposes key surface virulence factors 2) 107. Why is this most likely?: We agree with the referee and have rephrased this. Eldholm et al., 2010 indeed show that CbpD has muralytic activity, and that this activity requires functional CBD and SH3b domains.
3) 128. 'here we report': Wording has been adjusted accordingly 4) 137. Shouldn't "to" be "on"?: 'to' has been switched to 'on' to better clarify the meaning of the sentence 5) 163. This may be just a matter of taste but: the use of "essential" in this context may be misleading. We usually consider that if something essential is missing, the cell will be dead. Unless I misread the results, what is reported/detected here is that cells missing some identified genes are more susceptible to lytic killing, not absolutely "dead in the water.": The sentence has been edited to clarify that essential is meant in terms of more essential in protecting cells against lysis during competence 6) 164. Please define "top". The pathway shown here was constructed on basis of prior knowledge. These references have now been included.

9) 202. 10 TA operons?:
10 operons that contain the 14 sgRNAs found to become more essential in protecting cells against lysis during competence. Text has been edited to make this clear

10) 212. Is this repression or simply gene deletion?
This is a tricky point as the native genes are deleted and at an ectopic locus we have a copy under IPTG-inducible control. We changed the phrasing earlier from 'deplete' to 'not produce' to avoid potential confusion as well as changed repression to depletion in old line 212.

11) 211 (fig 5)
Please explain a bit more how these patterns should be understood. For example, what is the significance of ssbB expression very late into stationary phase? Also, where could a reader look to find the actual kinetics of its expression?: We decided to plot ssbB expression as area under the curve instead as kinetic curves so we are able to show many more data points in the same graph. Note that there is no stationary phase ssbB expression, the different bars represent different concentrations of inducer that is being used to control the expression of the gene of interest. For instance, if we look at the second plot, comCDE. Here comCDE is deleted at the native locus and is being expressed from Plac at the ZIP locus. In the absence of IPTG, ssbB-luc is not activated. With 5 uM of IPTG (the green bar) there is slight expression of ssbB-luc. At 1 mM of IPTG there is maximal ssbB-luc expression. Typical PssbB-luc curves have been published by us and others before, and we now cite this and made our figure legend more clear.

12) 225. Please explain the L/D scale. Would % lysed be a clearer metric?:
In principle, the used Y-axis is similar to a % lysis as suggested by the referee, as we normalize the fluorescence produced by dead cells (484nm emission) over the cell density (595nm). We do this as some depletion strains grow poorly. We have now better clarified this in the figure legends.

13) 392. How can a del-cps strain be encapsulated? Is there a typo here?:
This is a typo, it is meant to say 'unencapsulated'. Adjusted accordingly 14) 445. Is "subsequently" intended to mean "consequently"?: Yes, text has been adjusted accordingly 15) 456. As noted above, "essential" may be an oversimplification: Edited the text in a similar manner as above

16) 458. The operons do not "show an expected cost". Rather, it is interfering with these operons that creates a fitness cost:
Text has been adjusted to clarify this 17) l. 461. "to the contrary, inactivation of the comAB …. : Text has been adjusted accordingly 18) 463-5. The text seems confused here: Agreed and we have removed this section.

21) 848-853. Is the volume unit "ml" really intended?:
Many thanks for spotting this mistake. Indeed, it should be micro liter. It has been corrected in the revised manuscript.

22) 855. Note that "to an OD of 0.1" is redundant:
Removed this from the sentence 23) 857 and ff. Some steps have a temperature indicated, whilst others are not specified:

Adjusted text to add in missing temperatures
Reviewer 3, specific comments:

Lines 227-228: The authors have shown that ComM and TAs are important to maintain the integrity of cells during competence activation. To show that this process is in fact fratricide mediated, could the authors test whether these genes contribute to lysis in a fratricin-mutant (eg CbpD) background?:
In figure 2D and figure S6, these genes were tested for contribution to cell lysis in C + Y pH 6.8 media, which inhibits the development on natural competence and hence, inhibits the expression of cbpD. In figure S6J, the cps2A and psr mutants are tested for cell lysis in C + Y pH 6.8 media +/-CSP1. Hence, they were tested in the presence of fractricins upregulated during competence and in their absence when competence was not induced. Indeed, the referee is correct that depletion of the capsule makes cells more susceptible to lysis.

Lines
We have now clarified this in the revised manuscript. Indeed, recent work from the Filipe lab showed that unencapsulated pneumococci are more susceptible to autolysis by LytA and this has now been discussed. Fig 3C: In contradiction to the statement, the graph (6th panel in Fig 3C) does not show an increased protection (or less lysis) upon LytR overexpression?:

Lines 285-287 and
Actually, this panel (lytR -/Plac+) should be compared to the first panel (D39V) and it can be observed, especially when 0.25ug/ml CbpD is added, but less when 0.5ug/ml of CbpD was added, that there is additional protection. Most importantly however, both LytR and ComM overexpression concurrently provides better protection to lysis than when each protein is over expressed separately. We have clarified this now in the text to better reflect the data.
4. Line 312: It may be more accurate to state that ComM is "important" for LytR activity since increased WTA is produced upon LytR induction in a ComM mutant (Table S3): Agreed and we have rephrased this heading.

Lines 324-325: Fig 4B still shows the shift from a fold change of -1.8 to -0.2.
Good point. We note that addition of CSP1 would still overproduce LytR in the comM mutant thus explaining this partial shift to WTA. This has now been clarified in the revised manuscript.

Fig 4C: Why is there a reduction in WTA/LTA ratio upon overexpression of LytR in a ComM mutant?
Again a good point, and we agree this is indeed puzzling and in the revised manuscript we now note that currently we have no explanation for this observation.

Line 214: Can you normalize AUC RLU against the growth? To claim that the low RLU under low IPTG treatment is due to growth defect but not inadequate competence induction in order to rule out the possibility that the gene of interest is indeed regulating the competence development, show that after normalization against growth, there is no significant difference among different IPTG treatment groups.
This certainly could be done but does not make the data visualization easier. Any absence of a bar indicates lack of competence induction (e.g. compare comCDE NO IPTG to any other plot). This is now better clarified in the figure legend. Figure 3. Fig. 3C Unfortunately, deleting lytR is impossible due to the essential nature of LytR in the D39V genetic background. However, in Fig. 3C we did test for protection from CbpD induced cell lysis in a strain that was depleted for LytR and had normal expression of ComM. As expected, normal expression of ComM, even under low LytR levels, still provides low levels of protection against fratricide.

Line 269: In
10. Fig. 3C: Another issue with Fig. 3C is that it's not convincing to interpret the lysis results with the growth defect. Similar to Point 7 mentioned above, is there a way to normalize the lysis against growth?
In principle, the used Y-axis is normalized as suggested by the referee, as we normalize the fluorescence produced by dead cells (484nm emission) over the cell density (595nm). We do this as some depletion strains grow poorly. We have now better clarified this in the figure legend.

Line 370-372: What is the evidence to support this claim that the capsule is thinned by CbpD?
The referee is correct that there is no evidence in the literature to support this claim, so we have made it clear in the revised manuscript that this is our current hypothesis.
12. Line 409: Fig. 5: CbpD-dependent increased surface exposure of PspA and PspC is shown, but not any LytR-dependent effect, which made this part of the story off topic from the detailed analysis of TA. More experiments are needed to show the role of LytR and link to fratricins during this process.
Indeed the referee was correct that we did not show any LytR-dependent effect on surface exposure of PspA and PspC. Subsequently, we performed additional fluorescence microscopy experiments, testing LytR depletion and overexpression strains for PspA and PspC surface exposure. These new experiments found that overexpression of LytR amplified the difference in both PspA and PspC surface exposure after competence induction, while low levels of LytR expression abrogated this difference (new Fig. S7).
13. Line 409: Fig. 5: Data is insufficient to establish causality that the difference in adherence assay (Fig. 5C)  To provide further support for our hypothesis, we now also performed additional nasopharyngeal adherence assays using LytR overexpression and depletion strains.
These new experiments showed that indeed overexpression of LytR increased adherence to nasopharyngeal cells relative to wild type pneumococci, while depletion of LytR reduced adherence levels (new Fig. 3C). These results further support the hypothesis that increased CBP surface exposure due to competence induction impacts adherence to nasopharyngeal cells.
14. It exactly clear why the LytA-mediated shift from LTA to WTA effects CBPs since both forms of TA are contain phosphocholine? Is the hypothesis that WTA is more exposed than LTA? If so, evidence to support this concept would strengthen the proposed model.
This would indeed be in line with our model, but at this point we cannot provide experimental evidence to support this claim other than speculating that it is highly likely that CBPs that are bound to WTA are further away from the membrane than LTA-bound CBPs, and thus likely to be more surface exposed. It is now made more clear in the revision that this is based on speculation and prior assumptions regarding the positions of LTA and WTA within the pneumococcal cell envelope.
Minor Comments: 1. Line 49-51. It is confusing to state that Spn is a commensal and then describe it as a public health problem in the next line.
Text adjusted to clarify potential confusion 2. Line 154 and rest of the manuscript: 1 in CSP1 should not be in subscript.

Adjusted accordingly
4. Line 166: not "competent producing competitors", but "competence producing competitors" This information has now been added to the main text 'Fourteen sgRNAs targeting ten operons were significantly less abundant in condition IV, suggesting that they are essential or become more essential in protecting cells against lysis during competence ( Figures 1D and S2A, and Table S1; absolute log2FC>1, adjusted p-value<0.05).' and was already mentioned in the Data and code availability section (We tested against a log2FC of 1, with an alpha of 0.05).
6. Line 169: should be "either competence or TA synthesis" but not "both" 8. Line 188: This sentence is ambiguous. "Deletion of comM results in autolysis via CbpD production when competence is activated".
The text now reads "Deletion of comM results in autolysis when competence is activated, via CbpD production" to decrease potential ambiguity.

Line 194-195: didn't mention what's in grey.
Text edited to explain that in grey, genes involved in neither competence nor teichoic acid biosynthesis are shown.
10. Line 220: reference Fig. S5 instead of Fig. S6 Adjusted accordingly 11. Line 266-267: This sentence is confusing. Do you want to say as LytR is essential for normal growth, so you used lytR-/Plac-instead of a ∆lytR clean deletion to deplete for LytR? Also, in Fig. S6, you added 10µM IPTG to treat Plac-lytR-/+; but you didn't add IPTG to lytR-/Plac-in Fig. 2D. Is that because lytR-/Plac-doesn't require IPTG to remain viable as Plac-lytR-/+ does? The experimental setup is confusing until the reader reaches lines 270-6.
We have now better clarified this in the legend. Indeed, lytR is impossible to delete in the D39V genetic background hence the use of complementation strains. A correct citation has also been added.