The Mycobacterium tuberculosis PE15/PPE20 complex transports calcium across the outer membrane

  1. Vishant Boradia
  2. Andrew Frando
  3. Christoph Grundner

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Abstract

The mechanisms by which nutrients traverse the Mycobacterium tuberculosis ( Mtb ) outer membrane remain mostly unknown and, in the absence of classical porins, likely involve specialized transport systems. Calcium ions (Ca 2+ ) are an important nutrient and serve as a second messenger in eukaryotes, but whether bacteria have similar Ca 2+ signaling systems is not well understood. To understand the basis for Ca 2+ transport and signaling in Mtb , we determined Mtb’s transcriptional response to Ca 2+ . Overall, only few genes changed expression, suggesting a limited role of Ca 2+ as a transcriptional regulator. However, 2 of the most strongly down-regulated genes were the pe15 and ppe20 genes that code for members of a large family of proteins that localize to the outer membrane and comprise many intrinsically disordered proteins. PE15 and PPE20 formed a complex and PPE20 directly bound Ca 2+ . Ca 2+ -associated phenotypes such as increased ATP consumption and biofilm formation were reversed in a pe15/ppe20 knockout (KO) strain, suggesting a direct role in Ca 2+ homeostasis. To test whether the PE15/PPE20 complex has a role in Ca 2+ transport across the outer membrane, we created a fluorescence resonance energy transfer (FRET)-based Ca 2+ reporter strain. A pe15/ppe20 KO in the FRET background showed a specific and selective loss of Ca 2+ influx that was dependent on the presence of an intact outer cell wall. These data show that PE15/PPE20 form a Ca 2+ -binding protein complex that selectively imports Ca 2+ , show a distinct transport function for an intrinsically disordered protein, and support the emerging idea of a general family-wide role of PE/PPE proteins as idiosyncratic transporters across the outer membrane.

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  1. Version published to 10.1371/journal.pbio.3001906
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    Reply to the reviewers

    __Reply to the Reviewers __

    We thank the reviewers for their excellent suggestions and constructive comments. We now added new data on PE15/PPE20 binding to Ca2+, the PDIM status of mutant strains, additional controls, added to the discussion, added detail to the Methods, and provide all RNA-seq data. Please see replies to the comments in detail below:

    Reviewer 1:

    Major points

    1. Cellular localization:
    • “The authors do not describe the cellular fractionation method…”, “The authors show some Western blot data in Fig. S3, though the legend is superficial (abbreviations not explained) and the controls with markers for cellular localization appear to be lacking”. “Further, the authors do not prove that FLAG-tagged PE20 is functional.”

    We included a description of the fractionation method in Materials and Methods (lines 475-485). We also added detail to the legend of Fig. 4A to explain the abbreviations and controls used. The same cell fractions were used in Fig. 4A and Fig. S3A, as mentioned in the Figure S3 legend (“The same cell fractions as in Fig. 4A were used, see controls therein”). We know that the FLAG-tagged PPE20 is functional because the strain used in this experiment is the same we used for genetic complementation experiments in which FLAG-tagged PPE20 functionally complements ppe20 deletion in all three assays (ATP consumption, biofilm, Ca2+ influx, Fig.4 B,C,D,G).

    • “The authors should extend discussion part of the manuscript. Several proteomic studies.” “Did authors analyze culture filtrate fraction by Western?

    We thank the reviewer for the references and extended the Discussion to include results from existing proteomic studies on PE15/PPE20 (lines 229-234). We did not test for PE15/PPE20 in culture filtrate, and previous proteomic results are contradictory. Several PE/PPE proteins, including PE15/PPE20 have been detected in the cell wall and in the CFP, but not consistently. The functional significance of this dual localization is unclear.

    1. Is PE15/PPE20 a channel?
    • “PPE20 purified alone from the cytosol of E. coli?”

    We did not purify either protein by itself. As the reviewer correctly notes, PE/PPE proteins are refractory to individual purification. We clarified that we purified and used the complex for experiments even if only PPE20 is shown, as in Figure 3C,D, and E (Lines 124-127). See also Methods line 382 ff.

    • “…a positive control of a mutant that is indeed deficient in Mg2+ import (and thus showing a phenotype) is lacking.”

    Lacking a specific Mg2+ import mutant, and because it is a relatively minor point, we removed the statements about selectivity.

    1. Thermal melting assay
    • It is surprising to see that the thermal melting assays was done for PPE20 and PE15 as separately purified proteins.

    We co-purified PE15 and PPE20 for all biochemical experiments. We clarified that point (see also point 2 above).

    • “the thermal melting assay only seemed to give some results for PPE20 alone, and not for PE15”

    PE15 did not produce interpretable results in this assay, as mentioned in line 144. We clarified in the Fig. 3 legend that the complex was used although only PPE20 is detected by Western blot and shown in Figure 3C.

    • “…the results are counter-intuitive… How can the authors be sure that the presence of Ca2+ does not simply lead to more protein precipitation (via rather unspecific interactions) at elevated temperatures? Some positive controls with bona fide calcium binding protein in the same thermal melting setup would have helped to clarify this.”

    The effect of Ca2+ on PPE20 is somewhat counterintuitive, although not unprecedented. Proteins can be stabilized or destabilized by ligand binding, and a recent proteome-wide study on the basis of thermal shift analysis showed that ~17% of proteins were destabilized by ligand (ATP). For a channel in particular, ligand binding might be expected to be coupled to protein relaxation in the process of channel opening, which could well translate to lower thermal stability. We added the positive control showing the behavior of a known Ca2+ binding protein (new Fig. S2A). In addition, we included a negative control showing that Ca2+ does not generally increase protein denaturation (Fig. S2B). We think that this control addresses the reviewer’s concern more directly.

    • If the authors want to stick to their claims regarding Ca2+ binding to PE15/PPE20, they have to perform additional assays (e.g. equilibrium dialysis or ITC) with the entire PE15/PPE20 complex. Further, they have to show that PE15/PPE20 forms a proper oligomeric protein that is membrane bound and reasonably behaved on size exclusion chromatography, when expressed in and purified from E. coli.

    Detecting Ca2+ binding to proteins is not trivial, and we thank the reviewer for suggesting equilibrium dialysis as another, orthogonal assay. We now show an equilibrium dialysis experiment that confirms Ca2+ binding by the PE15/PPE20 complex. Please see the new Fig. 3F. and G. and lines 146-152 (Results) and 429-443 (Methods).

    The PE/PPE proteins are generally difficult to express and purify recombinantly, likely due to the typically large unstructured regions. Also, the yield of PE15/PPE20 when expressed in E. coli was very low so that we were not able to detect the complex by SEC. However, data in Fig. 3 conclusively show that PE15 and PPE20 bind.

    1. RNA-seq data
    • The authors should include a table with all other identified genes that are potentially involved in calcium homeostasis

    We provided all other significant differentially expressed genes in the new Table S1.

    Minor points:

    1. “what is the binding affinity of the Ca sensor?”

    We added the Ca2+ binding affinity of Twitch-2B (KD: 200nM) in line 176.

    1. Figure 4D: “one would expect a drop in FRET signal after EGTA addition… Can the authors explain?”

    We do see a clear drop in FRET signal after EGTA addition, in particular in 7H9 medium (black versus red line, Fig. 5B). Given the high affinity of Twitch-2B for Ca2+ (200nM), however, it is not surprising that the drop is not more pronounced, as intracellular Ca2+ is expected to be tightly bound to Twitch.

    1. The experiments showing outer membrane localization of PE15/PPE20 are very important, but results of these experiments (western-blot and FRET) are shown in supplementary figures. They should be transferred/integrated into the main Figures.

    We agree and moved Figure S3A to the main Figures as Figure 4A.

    1. Line 166: the authors claim that the assay did not work in 7H9 due to low Ca2+ concentration in this medium. Why did the authors not just add a bit more calcium to show whether this claim holds true?

    7H9 is not a suitable medium for these experiments because the baseline Ca2+ concentration is too high, not too low (see Fig. 5B, grey versus black line). Adding more Ca2+ to 7H9 medium resulted in precipitation, probably due to its interaction with phosphates. Our use of “low” in this context was confusing, we changed the wording of this sentence (line 180-181).

    1. Line 183: more detailed description on cellular fractionation and subsequent anti-FLAG Western needed here.

    We added more detail in the Materials section (lines 475 ff).

    Reviewer 2:

    • A major concern regarding the importance of the data: there are considerable technical challenges in generating Ca2+ depleted media. This is clear in that M. tuberculosis seems to be unaffected by Ca2+ in the medium - similar growth seems in Ca2+-free media to media with up to 10mM Ca2+ (Fig. S1). This raises a concern about the physiological relevance of the data (mammalian cells have intracellular Ca2+ of 0.01-0.1mM, extracellular free Ca2+ is around 1mM).

    If we correctly understand this comment, the reviewer is unconvinced that we fully and reproducibly depleted Ca2+ from medium based on a lack of an effect of Ca2+ on in vitro growth. We tested for baseline Ca2+ levels and depletion in media by inductively coupled plasma optical emission spectrometry and added these data showing precise quantitation of Ca2+ in medium (see new Fig. S1B).

    • The role of PE15/PPE20 in Ca2+ acquisition may be clearer if the authors ensure that the PDIM layer is intact. Specifically, there is a technical issue: The authors use Tween80 as a detergent. Tween-80 partially strips the outer cell wall of M. tuberculosis resulting in shedding of PDIM and PE/PPE proteins. Tyloxapol is a somewhat milder detergent. Some of the experiments would possibly show clearer phenotypes by use of Tyloxapol.

    We share the concern about PDIM, as PDIM loss is common in in vitro culture. We analyzed the total lipids by thin layer chromatography and confirmed the presence of PDIM in all three strains (Fig S3C, lines 198-201). We repeated experiments with Tyloxapol and did not see differences to Tween-80. We nonetheless now show the Tyloxapol data (Fig 5D).

    • The authors could increase the impact of their work be exploring the role of PE15/PPE20 during pathogenesis of resting versus activated bone marrow macrophages where Ca2+ fluxes of the host cell play a role in host responses.

    We agree. In vivo or macrophage experiments are a logical next step to fully characterize the function of PE15/PPE20, but we think it is beyond the scope of this manuscript. The main contribution of this paper is the identification of channel function of a PE/PPE protein pair that extends the novel channel paradigm for these proteins. These data support that transport might indeed be a shared function of the entire PE/PPE family with 169 members.

    Minor:

    • The authors should consider citing Sharma et al (2021)

    We cited the paper.

    • Are there Ca2+ binding motifs in PPE20?

    We did not detect canonical Ca2+ binding motifs in PPE20.

    • RNAseq data may need to be deposited in a public database.

    RNA-seq data have been deposited to NCBI - GEO accession GSE214266

    Link: https://urldefense.com/v3/https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE214266;!!NuzbfyPwt6ZyPHQ!tCf4MS_HRKJFn6qV2orkDAkXTWvx9IIU11fAV7TguYE2ietoMBpBgRC7rvfnM9bsoiVdIvDBUHdPmHZliDP2o5sRZR2ziK4$

    Token: cvmhakcgbpmbfuz

    • In its current state, the work is somewhat incremental

    The function of the large PE/PPE protein family of Mtb has been one of the most longstanding and perplexing puzzles in Mtb biology. For more than 20 years, speculation about their potential role, for example in antigenic variation, abounded but no conclusive evidence for this or another shared function emerged. A recent landmark paper then conclusively showed that a subset of the PE/PPE proteins function as nutrient channels (Wang et al., Science 2020). However, whether transporter function is a general function of the family of 169 PE/PPE proteins remains untested. Our PE/PPE pair is associated with a different type VII secretion system (Esx-3) and belongs to a different subfamily than the previous examples, suggesting a shared function across families and perhaps even all of these proteins. Given the intense interest and many false leads that have plagued the identification of PE/PPE function in the last 20 years, the difficulty of working with them biochemically, as well as the almost complete absence of understanding of Ca2+ homeostasis in Mtb, we do not consider our work incremental.

    Reviewer 3

    • My only slight concern is the meaning attached to the "biofilm" assays. It is never very clear to me that this is anything more than formation of a surface pellicle and general hydrophobicity of the mycobacterial cells.

    We fully agree that Mtb biofilms remain poorly defined. However, the term biofilm as used in our study has already found its way into the literature and we would rather not cause confusion by calling the same phenomenon by a different name. Whatever the term used, we do not suggest any other relevance other than it being a Ca2+-dependent phenotype that serves as one of several tests to parse PE15/PPE20’s role in Ca2+ homeostasis.

    Cross-consultation comments:

    • We agree with the concerns of reviewer#2 that the role of PDIM and use of detergent should be looked at more closely.

    We tested the roles of PDIM and detergent, see reviewer 2.

    • Likewise, the paper would strongly benefit from some further insights into the potential physiological role of PPE20/PE15 in calcium homeostasis.

    We show PE15/PPE20 function in the transport of Ca2+ and the first Ca2+-related cellular phenotypes in Mtb. Testing the role of the complex in an infection model is outside of the scope of this manuscript and mouse infection experiments would take many months and would likely be intractable because of the expected extensive redundancy among the 169 PE/PPE proteins.

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    Referee #3

    Evidence, reproducibility and clarity

    Review of Boradia et al, "Calcium transport by the Mycobacterium tuberculosis PE15/PPE20 proteins" This manuscript describes studies aimed at understanding the role of calcium in the pathogenesis of tuberculosis. The authors begin by showing that, analogous to the situation in other bacteria, ATP levels are directly (and rather dramatically) affected by extracellular calcium levels. The authors then look at the effect on "biofilm formation" and, again analogous to other bacteria, find a link. The authors then perform RNAseq on bacterial cells with and without 1mM Ca++ and identify a pair of genes that is strongly downregulated by calcium sufficiency. These genes are PE/PPE family members which have been recently associated with channel formation in the mycomembrane to allow transport of small molecule solutes across the outer cell envelope. The authors show these proteins are associated in a complex by reciprocal pull-down experiments in tagged proteins and show directly that they bind calcium by a thermal stability change of this complex in the presence of calcium. Finally, they show, using a calcium sensitive FRET reporter expressed in Mtb, that these two proteins allow calcium influx and that such an influx is blocked in a strain where they have been deleted.

    Overall, the study is excellent and convincingly establishes the transport function of another pair of PE/PPE proteins. My only concern with this is that they stop just short of delving into the actual infection biology of calcium, but I suppose that will be next. The tools they developed in this study, specifically the knockout strain and the FRET reporter, put them in a strong position to explore the role of calcium during growth in macrophages and other in vivo studies that are surely planned.

    My only slight concern is the meaning attached to the "biofilm" assays. It is never very clear to me that this is anything more than formation of a surface pellicle and general hydrophobicity of the mycobacterial cells. I wonder if the presence of calcium alters the aggregation state of the bacilli and or affects the surface in some more subtle manner. I am not convinced that the word "biofilm" as it is used commonly in other bacteria, has anything to do with the physical properties that are being observed in the case of Mtb.

    Significance

    The manuscript clearly establishes that this pair of PE/PPE proteins plays a direct role in calcium transport in MTB and provides several useful tools to begin to understand the role of calcium in TB pathogenesis. The work is outstanding and novel.

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    Referee #2

    Evidence, reproducibility and clarity

    The authors demonstrate that M. tuberculosis responds to increasing Ca2+ concentrations by increasing ATP levels as well as increased ability to form biofilms. Culturing of M. tuberculosis with Ca2+ results in downregulation of pe15/ppe20. The proteins are recombinantly expressed and the results show that PE15 and PPE20 form a complex. In addition, PPE20 seems to be destabilized by Ca2+ suggesting that it interacts with this metal ion. A pe15/ppe20 knockout shows lower levels of ATP increase upon incubation with Ca2+ although the differences with would-type are very modest. Similarly, the knockout shows an impaired ability to form biofilms at 1mM and 10mM Ca2+. Finally, the authors make a FRET reporter of intracellular Ca2+ concentrations based on the Twitch system which nicely shows that intracellular Ca2+ levels are lower in the knockout mutant.

    Overall, the data suggest that PE15/PPE20 are involved in Ca2+ uptake which contributes to our evolving understanding of the role of the different PE/PPE proteins in nutrient acquisition. The highlight of the paper is the Twitch bioreporter for Ca2+ which could be useful in exploring the role of Ca2+ in mycobacteria.

    A major concern regarding the importance of the data: there are considerable technical challenges in generating Ca2+ depleted media. This is clear in that M. tuberculosis seems to be unaffected by Ca2+ in the medium - similar growth seems in Ca2+-free media to media with up to 10mM Ca2+ (Fig. S1). This raises a concern about the physiological relevance of the data (mammalian cells have intracellular Ca2+ of 0.01-0.1mM, extracellular free Ca2+ is around 1mM). The role of PE15/PPE20 in Ca2+ acquisition may be clearer if the authors ensure that the PDIM layer is intact. Specifically, there is a technical issue: The authors use Tween80 as a detergent. Tween-80 partially strips the outer cell wall of M. tuberculosis resulting in shedding of PDIM and PE/PPE proteins. Tyloxapol is a somewhat milder detergent. Some of the experiments would possibly show clearer phenotypes by use of Tyloxapol. In experiments where clumping is not a concern (ATP measurement), the cells can be pre-grown as indicated but then transferred to the multiwell plates in detergent-free media. At the time of processing of the cells for readout of, for example ATP, detergent can be used as needed. The authors could increase the impact of their work be exploring the role of PE15/PPE20 during pathogenesis of resting versus activated bone marrow macrophages where Ca2+ fluxes of the host cell play a role in host responses.

    Minor:

    The authors should consider citing Sharma et al (2021): PGRS Domain of Rv0297 of Mycobacterium tuberculosis functions in A Calcium Dependent Manner

    Are there Ca2+ binding motifs in PPE20?

    RNAseq data may need to be deposited in a public database.

    Significance

    In its current state, this work is somewhat incremental: the authors have provided data that suggest that PE15/PP20 are involved in Ca2+ uptake (data could be strengthened as suggested above). The physiological relevance of the PE15/PPE20 system remains unclear - no data on its role in pathogenesis.

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    Referee #1

    Evidence, reproducibility and clarity

    PE/PPE proteins build up 10% of genome of Mtb, but the function of these proteins is only currently investigated in more detail. Recent studies show the involvement of individual PE/PPE proteins in the transport of nutrients, and more data supporting this functional role are about to emerge. Using RNA-seq, Boradia et al identified the pe15/ppe20 genes to be downregulated in response to calcium exposure. Purified PPE20 (but not PE15) appear to bind calcium in a thermal stability assay (though this claim needs further experimental support). The authors generated a Mtb pe15/ppe20 knockout strain and convincingly show in three types of assays (ATP levels, biofilm formation and lower signal in FRET measurements corresponding to lower calcium concentrations compared to the wild type strain) that the PE15/PPE20 proteins are involved in cellular calcium import, but do not appear to import magnesium. All phenotypes could be restored to the behavior of wildtype Mtb by complementing the KO strain with pe15/ppe20.

    The manuscript is clearly written and easy to follow. The authors combined molecular biology (RNA-seq), biochemistry (proteins purification), biophysics (FRET) and microbiology (knockout generation, in vivo measurements) to reach their conclusions. Overall, the study reports novel and interesting data and as such is of high interest for the mycobacterial research community. However, some of the claims have a rather experimental basis, and thus the study needs to be strengthened with further experiments (or statements have to be removed) as outlined below.

    Major points:

    1. Cellular localization of PE15/PPE20.

    The authors do not describe the cellular fractionation method they applied (no mentioning of cellular localization experiments in materials and methods). The same applies to the main text (very superficial description). The authors show some Western blot data in Fig. S3, though the legend is superficial (abbreviations not explained) and the controls with markers for cellular localization appear to be lacking. Further, the authors do not prove that the FLAG-tagged PPE20 is functional.

    The authors should extend discussion part of the manuscript. Several proteomic studies did not identify PE15 or PPE20 in the cell wall - doi: 10.1021/pr1005873, doi: 10.1091/mbc.E04-04-0329. At the same time PE15 (but not PPE20) is membrane or membrane-associated protein according to this work: doi: 10.1186/1471-2180-10-132. Quite recent work (https://doi.org/10.1073/pnas.1523321113) showed that PE15/PPE20 are secreted substrates of ESX-3 and these proteins have been found in the culture filtrate. Did authors analyze culture filtrate fraction by Western blotting?

    1. Is PE15/PPE20 a channel?

    A major claim of the authors is that PE15/PPE20 forms a (specific) channel for Ca2+ and not a porin-like protein that is permeable to a large set of solutes. However, this claim has its main experimental backing that PPE20 (purified alone from the cytosol of E. coli?) binds to Calcium in a (rather weirdly looking) "thermal melting assay" (further comments on these assays, see below). The second experiment supporting this idea is a lack of difference between wt and KO strain of Mtb in an assay that should report Mg2+ transport deficiency (Fig. S3). But here, a positive control of a mutant that is indeed deficient in Mg2+ import (and thus showing a phenotype) is lacking. In conclusion, the experimental basis on the grounds of which the authors claim PE15/PPE20 to be a specific Mg2+ channel is weak. On the other hand, the functional data clearly show a link between PE15/PPE20 and calcium uptake: Hence the data are solid enough to claim that PE15/PPE20 facilitates Ca2+ transport across the mycomembrane.

    1. Thermal melting assay.

    It is surprising to see that the thermal melting assays was done for PPE20 and PE15 as separately purified proteins. How did you purify PPE20 alone for this assay? It is broadly accepted for PE/PPE proteins that they only can be purified as pairs, including for PE15/PPE20 (https://doi.org/10.1073/pnas.0602606103). As for the cellular localization, the method section falls short in providing relevant information on how PPE20 and PE15 were purified in separate forms (it states they were co-expressed using a pETDuet vector). Further, the thermal melting assay only seemed to give some results for PPE20 alone, and not for PE15. There is no mentioning of the PE15/PPE20 complex in this assay. Further, the results are counter-intuitive, as Ca2+ addition leads to more precipitation at higher temperatures (and it does seem to weaken the stability of PPE20 instead of stabilizing it). How can the authors be sure that the presence of Ca2+ does not simply lead to more protein precipitation (via rather unspecific interactions) at elevated temperatures? Some positive controls with bona fide calcium binding protein in the same thermal melting setup would have helped to clarify this.

    If the authors want to stick to their claims regarding Ca2+ binding to PE15/PPE20, they have to perform additional assays (e.g. equilibrium dialysis or ITC) with the entire PE15/PPE20 complex. Further, they have to show that PE15/PPE20 forms a proper oligomeric protein that is membrane bound and reasonably behaved on size exclusion chromatography, when expressed in and purified from E. coli. As it is doubtful that the authors can meet such quality standards, I would recommend to remove all statements regarding Ca2+ binding to PPE20 from the manuscript, as the underlying experiments are of poor quality.

    1. RNA-seq data

    The authors should include a table with all other identified genes that are potentially involved in calcium homeostasis. This is of interest because the KO strain is still capable of calcium import, hence other Ca2+ transport systems likely exist.

    Minor comments:

    1. FRET experiments What is the binding affinity of the sensor for calcium?
    2. Figure 4D: one would expect a drop in FRET signal after EGTA addition, because this reverts the Ca2+ gradient from out-to-in (thus facilitating calcium flow into the cells) to in-to-out (EGTA actually acting as a sink into which all Ca2+ (also the one from within the cell) would flow). Can the authors explain?
    3. The experiments showing outer membrane localization of PE15/PPE20 are very important, but results of these experiments (western-blot and FRET) are shown in supplementary figures. They should be transferred/integrated into the main Figures.
    4. Line 166: the authors claim that the assay did not work in 7H9 due to low Ca2+ concentration in this medium. Why did the authors not just add a bit more calcium to show whether this claim holds true?
    5. Line 183: more detailed description on cellular fractionation and subsequent anti-FLAG Western needed here.

    Referees cross-commenting

    We agree with the concerns of reviewer#2 that the role of PDIM and use of detergent should be looked at more closely.

    Likewise, the paper would strongly benefit from some further insights into the potential physiological role of PPE20/PE15 in calcium homeostasis.

    Significance

    Slow-growing mycobacteria like Mtb lack porins. Therefore, it is not clear how nutrients can be transported through the outer membrane. More and more data hint on PE/PPE protein family that can fulfill this function (Wang et al., Science 367, 1147-1151 (2020)). In the current work, the authors show that PE15/PPE20 are involved in calcium transport in Mtb. Mtb is a difficult model organism to work with because of its pathogenicity and slow rate of growth. Therefore, any information on nutrients transport in Mtb is highly appreciable.

    The RNA-seq experiments as well as the genetic/functional experiments clearly show that PE15/PPE20 facilitates calcium import in Mtb. The corresponding sections and figures are convincing.

    The experimental data attempting to show PE15/PPE20's cellular localization and its interaction with Ca2+ are currently weak, and need to be strengthened.

  6. Version published to 10.1101/2022.05.30.494056v1 on bioRxiv