MicroRNA-27a controls the intracellular survival of Mycobacterium tuberculosis by regulating calcium-associated autophagy

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) kills millions every year, and there is urgent need to develop novel anti-TB agents due to the fast-growing of drug-resistant TB. Although autophagy regulates the intracellular survival of Mtb, the role of calcium (Ca2+) signaling in modulating autophagy during Mtb infection remains largely unknown. Here, we show that microRNA miR-27a is abundantly expressed in active TB patients, Mtb-infected mice and macrophages. The target of miR-27a is the ER-located Ca2+ transporter CACNA2D3. Targeting of this transporter leads to the downregulation of Ca2+ signaling, thus inhibiting autophagosome formation and promoting the intracellular survival of Mtb. Mice lacking of miR-27a and mice treated with an antagomir to miR-27a are more resistant to Mtb infection. Our findings reveal a strategy for Mtb to increase intracellular survival by manipulating the Ca2+-associated autophagy, and may also support the development of host-directed anti-TB therapeutic approaches.

Also, the authors might examine the role of mir27a in repression of PINK1, a regulator of mitochondrial autophagy and a known target of mir27a (Molecular Neurodegeneration 2016 11:55). This is because Parkin, the partner of PINK1 in mitophagy, has also been suggested to be involved in stimulating autophagy of Mtb.
Major points 1) How many founders were used for the mir27a-/-mice? Have they been backcrossed to exclude off-target effects of the gRNA in the first generations? This information is needed to interpret properly the validity of the data using this model. Similarly, information on the strain background of the Cacna2d3 mice, the breeding program to maintain homozygotes and what the strain background of the wild-type controls used to compare these homozygotes against is required. Also, what is the targeted gene mutation in Canc2d3 -what does it do the the function of this gene? 2) Lines 135-138. Experiments with pleiotropic autophagy blocking drugs should be performed with, for example, Atg5-/-macrophages to ensure the apparent redundancy with mir27a of autophagy is really autophagy and not some other target of these drugs 3) Proper statistical treatment of in vivo infection (CFU assay) experiments is missing from figures throughout. This is a presumed oversight? Statistical thresholds used for these experiments are indeed given in the figure legends… 4) Figure 1 -it does appear that autophagic flux is increased with mir27a ablation. It is not possible to say that this is due to increased autophagosome maturation per se. A true flux assay, biochemically (LC3B-II blot) or by immunofluorescence (tandem LC3 reporter), such as a time course in the presence versus absence of chloroquine or bafilomycin is required to resolve this. This is worth doing as the regulation of autophagy by mir27a-/-is a fundamental observation in this study that requires complete characterisation. Similar comments apply to the later flux assays performed where Cacna2d3 levels are manipulated. 5) In addition to point 4, the authors should exclude that the association of Mtb with autophagosomes (albeit forming at a reduced rate due to mir27a) is not disrupted by mir27a (i.e. abundance of Mtb-positive autophagosomes should reduce proportionally with overall reduction in autophagosomes). 6) It would be useful to present controls for the efficacy of mir-27a antagomir on mir27 levels/function in the in vitro and in vivo experiments (i.e. known rather than novel mir27 targets described here or other readout?). This could then enable the reader to conclude that the effects of the antagomir are likely to be "on-target" effects. 7) In the TEM analysis of Cac3m/m macrophages (lines 237), are these bacteria unincorporated into autophagosomes? Is there a difference observable between these and the proportion of Mtb +ve autophagosomes in the TEM from wild-type mice? 8) Figure 4i -Mtb infection perturbs calcium levels within seconds, as shown, but effects on autophagy and viability are on the time scale of hours. What is the effect on calcium levels at such later time points where evidence of autophagy regulation can actually be obtained? 9) Figure 5. The pathology of lungs in the infection model is presented largely without any quantification. Are these changes quantitatively significant between the genotypes? Minor points 1) Define PBMCs first time acronym is used (line 94) 2) Why did the authors choose to focus on mir27a after the initial expression analyses (Figure 1ac)? An explanatory note to this effect would be good.
3) Lines 111-112. Use of these chemical inhibitors does not conclusively demonstrate a TLR2/ERK/NfkappaB signaling pathway. This should be acknowledged in text. 4) Lines 118-124. For clarity, the authors should be explicit about how they conclude the initial infection/phagocytosis of Mtb is unaffected but the subsequent survival is affected (i.e. analysis of different time points within the data). 5) Line 135 -"influx" is not correct, "flux" is the correct term. 6) Please check that panels 4a and 4b are in the correct order and referenced so in the text and Reviewer #3: Remarks to the Author: This manuscript describes the regulation of autophagy by Mycobacterium tuberculosis via microRNA (miRNA) miR-27a and an endoplasmic reticulum calcium transporter. This is a novel finding providing new insight to the intracellular survival of the tubercule bacillus. The work is exciting because M. tuberculosis encodes a number of proteins known to bind calcium. The authors provide a compelling story that convincingly shows this novel regulatory pathway. I found the work to be comprehensive and very well done. I recommend the acceptance of this paper.

Reviewer #1 (Remarks to the Author):
Liu et al reported that miR-27a is highly expressed in active TB patients, in the lung of Mycobacterium tuberculosis (Mtb)-infected mice, and in Mtb-infected macrophages. They showed that miR-27a inhibits autophagy by suppressing the expression of its target gene CACNA2D3 (Cac3), a calcium transporter located on the ER. By generating and analyzing miR-27a -/and Cac3 mutant mice (Cac3m/m), as well as their macrophages, they showed that miR-27 facilitates Mtb survival, while Cac3 promotes host control of Mtb. Furthermore, the authors showed that in Cac3m/m mice and macrophages, the effect of miR-27a inhibitors and mimics on autophagy and Mtb is abrogated, suggesting that miR-27a controls autophagy and Mtb survival through Cac3 and calcium signaling. The finding that Mtb induces miR-27a expression to facilitate the former's escape from autophagy-mediated killing by suppressing Cac3-mediated calcium signaling, which was previously shown to promote autophagy, is novel and interesting. However, there are a few concerns that need to be addressed: We thank the reviewer for appreciating the novelty of our current work, and will address the concerns as following.
1. To what degree do Cac3 and calcium signaling mediate the effect of miR-27a on autophagy and Mtb survival? An easy genetic test is to generate miR-27a-/-;Cac3m/+ and miR-27a-/-;Cac3m/m mice and to assess whether restoring Cac3 expression to WT levels is sufficient to negate the effects of miR-27a-deficiency. A few results presented in this manuscript suggest that the functional importance of Cac3 and calcium signaling in mediating miR-27a effect may be more modest than the authors would like to suggest: We appreciate the reviewer's comments. Our current data showed that the effect of the miR-27a mimic or inhibitor on the survival of Mtb or the formation of LC3 puncta is observed in wild type (WT) macrophages, but not in Cac3 PB/PB macrophages ( Fig.  6a-d, Supplementary Fig. 13a, b), suggesting that Cacna2d3 may mediate the miR-27a's effect in regulating the process of Mtb infection.
However, we fully agree with the reviewer that generation of miR-27a -/-Cac3 PB/PB mice to assess whether restoring Cacna2d3 expression to WT levels is sufficient to negate the effects of miR-27a-deficiency would be much helpful to strengthen our finding that Cacna2d3 mediates the effect of miR-27a on autophagy and Mtb survival. We have tried very hard to generate miR-27a -/-Cac3 PB/PB mice for a long time, but haven't got it so far, probably due to the low fertility. a. Fig. 3k,l: while Cac3 mRNA and protein are expressed at very low levels in WT lung and macrophages, they are drastically increased in miR-27a -/lung and microphages. Somehow this drastic increase in Cac3 expression did not translate into a strong effect on calcium signaling (Fig. 6f). This is surprising considering that calcium signaling is completely abolished in Cac3 m/m macrophages (Fig. 4i).
One has to argue that the trace amount of Cac3 in WT macrophages is sufficient for its function and any further increase in the cellular concentration of Cac3 protein doesn't significantly enhance calcium signaling. If this is true, pathways other than Cac3 and calcium must be invoked to explain the significant effect of miR-27a deficiency on autophagy (Fig. 1j, k, l) and Mtb control (Fig. 2). Thank the reviewer for the illuminating question. Cacna2d3 encodes a regulatory α 2 δ subunit of voltage gated calcium channels, which is required for the assembly of the channel on the membrane. Cacna2d3's effect on calcium signaling may be modest, most likely due to the loose association of α 2 δ subunit with the channel (Cassidy et  al., 2014, Zamponi et al., 2015), and a possible saturation of channel assembly on the membrane. That may explain why increase in the cellular concentration of Cacna2d3 protein only lead to a ~ 2-fold increase of calcium signaling (Fig. 6e, f). Furthermore, we agree with the reviewer's point that miR-27a may have multiple targets other than 2. As miR-27a exists in the miR-23a/27a/24-2 cluster, deletion of miR-27a may affect the expression of the other two miRNAs in the cluster, i.e. miR-23a and miR-24. The expression levels of miR-23a and miR-24 should be examined in miR-27a-/-lung and macrophages, and compared with their WT counterparts. As per the reviewer's suggestion, we have analyzed the expression of miR-23a and miR-24 in the macrophages isolated from miR-27a -/mice, and compared them with their WT counterparts. The data demonstrated that the expression levels of miR-23a and miR-24 show no significant difference between miR-27a -/and WT macrophages. We have added this part in the main text (Supplementary Fig. 2d).

Fig. 4c: TEM analysis needs quantification.
As per the reviewer's suggestion, we have provided the quantification of TEM analysis in the revised manuscript (Supplementary Fig. 8e).

Fig. 5d: The immunohistochemistry of LC3B is hard to interpret. Does blue staining indicate LC3B? If so, why did the Cac3m/m lung show more LC3B staining?
The brown staining indicated LC3B. According to our results, there is less LC3B staining in the lung tissues of Cac3 PB/PB mice as compared to the WT mice. As per the reviewer's suggestion, we have added this part to the figure legend of revised manuscript. Fig. 6e: is the color code labeled correctly? The text and figure do not match. We thank the reviewer for the correction. The labels of the color code have been corrected in the revised manuscript (Fig. 6e).

Not much is known about the regulation of calcium-dependent autophagy mechanisms in either normal physiology or pathophysiology. Liu et al show that Mycobacterium tuberculosis (Mtb) infection induces a miRNA (mir-27a) in human and mouse cells. Via a novel target, an ER-resident calcium channel containing the CACNA2D3 polypeptide, this results in suppression of anti-microbial autophagy in cell models and in mouse models of Mtb infection. This is a very interesting finding with likely clinical relevance too, with mir-27a upregulated in human cases of tuberculosis and antagomirs of mir-27a diminishing infection burden in mouse models of Mtb infection. Generally,
speaking the manuscript is well written with little redundancy with existing published findings. Most experiments are well-designed leading to mostly robust conclusions. The methods are mostly well-documented, enabling reproducibility. We thank the reviewer for pertinent comments and appreciating for the novelty and quality of our study.
There are nonetheless a number of specific instances where these standards are not reached (see specific points below). These should be addressed prior to publication.
As per the reviewer's suggestion, we have addressed the reviewer's concern as described below.
Also, the authors might examine the role of mir27a in repression of PINK1, a regulator of mitochondrial autophagy and a known target of mir27a (Molecular Neurodegeneration 2016 11:55). This is because Parkin, the partner of PINK1 in mitophagy, has also been suggested to be involved in stimulating autophagy of Mtb.
As per the reviewer's suggestion, we have analyzed the effect of Pink1, a regulator of mitochondrial autophagy and a known target of miR-27a (Kim et al., 2016), on intracellular survival of Mtb in WT and miR-27a -/macrophages. The data showed that inhibition of Pink1 by RNAi significantly decrease the intracellular survival of Mtb in both of WT and miR-27a -/macrophages as determined by CFU assay, indicating Pink1 as a positive regulator of Mtb survival. Therefore, the enhanced effect of miR-27a on the intracellular survival of Mtb is unlikely through downregulating Pink1. We have added this part into the main text ( Supplementary  Fig. 13d, e).

Major points 1)
How many founders were used for the mir27a-/-mice? Have they been backcrossed to exclude off-target effects of the gRNA in the first generations? This information is needed to interpret properly the validity of the data using this model. Similarly, information on the strain background of the Cacna2d3 mice, the breeding program to maintain homozygotes and what the strain background of the wild-type controls used to compare these homozygotes against is required. Also, what is the targeted gene mutation in Canc2d3 -what does it do the function of this gene? Thank the reviewer for the question. We have obtained 3 F1 founders for the miR-27a -/mice, and chosen one of them to backcross to the WT mice more than 5 generations to maintain the strain. The information was added to the methods section.
Thank the reviewer for the question. The Cacna2d3 mutant mice were generated using piggyback (PB) transposon in FVB strains, and were gifted by Prof. Xiaohui Wu from Fudan University (Ding et al., 2005, 2014). Briefly, PB transposon was inserted into the intron between exon 27 and exon 28 of the Cacna2d3 gene loci, and the expression of Cacna2d3 is subsequently disrupted (Supplementary Fig. 8b) To be more precisely, we have revised the description of the Cacna2d3 mutant mice as Cac3 PB/PB in the whole manuscript.

2)
Lines 135-138. Experiments with pleiotropic autophagy blocking drugs should be performed with, for example, Atg5 -/macrophages to ensure the apparent redundancy with mir27a of autophagy is really autophagy and not some other target of these drugs. As per the reviewer's suggestion, we have applied Atg5 -/macrophages and confirmed that the altered viability effect of miR-27a mimic or inhibitor on the Mtb infection is truly through autophagy (Supplementary Fig. 3c, d).

3) Proper statistical treatment of in vivo infection (CFU assay) experiments is missing from figures throughout. This is a presumed oversight? Statistical thresholds used for these experiments are indeed given in the figure legends…
As per the reviewer's suggestion, we have added the statistics treatment and included the thresholds of in vivo infection experiments into the figures (Fig. 2c, 2f, 5c and  6m). Figure 1 -it does appear that autophagic flux is increased with mir27a ablation. It is not possible to say that this is due to increased autophagosome maturation per se. A true flux assay, biochemically (LC3B-II blot) or by immunofluorescence (tandem LC3 reporter), such as a time course in the presence versus absence of chloroquine or bafilomycin is required to resolve this. This is worth doing as the regulation of autophagy by mir27a-/-is a fundamental observation in this study that requires complete characterisation. Similar comments apply to the later flux assays performed where Cacna2d3 levels are manipulated.

4)
As per the reviewer's suggestion, we have examined the mRFP-GFP-LC3 reporter pattern in the presence versus absence of chloroquine to carefully determine the effect of mir-27a on autophagy flux. The results showed that in the presence of chloroquine (CQ) (Ouimet et al., 2016), deficiency of miR-27a dramatically increase autophagosome formation and LC3 activation, suggesting that the increased autophagic flux with miR-27a ablation is due to increased autophagosome maturation per se. We have added this part into the main text (Supplementary Fig. 4a-c). Also, our results showed that in the presence of CQ, deficiency of Cacna2d3 lead to dramatically decreased LC3-II amount during Mtb infection. (Supplementary Fig.  8f) 5) In addition to point 4, the authors should exclude that the association of Mtb with autophagosomes (albeit forming at a reduced rate due to mir27a) is not disrupted by mir27a (i.e. abundance of Mtb-positive autophagosomes should reduce proportionally with overall reduction in autophagosomes). According to our results, miR-27a was shown to suppress both autophagosome formation and the Mtb-autophagosome co-localization, but overall reduction in autophagosomes appears to be very close to Mtb-positive autophagosomes (Fig. 1i,  Supplementary Fig. 14d). These results suggested that the increased Mtb-positive autophagosomes in miR-27a -/macrophages may at least partially result from the enhanced autophagosome formation.
6) It would be useful to present controls for the efficacy of mir-27a antagomir on mir27 levels/function in the in vitro and in vivo experiments (i.e. known rather than novel mir27 targets described here or other readout?). This could then enable the reader to conclude that the effects of the antagomir are likely to be "on-target" effects. As per the reviewer's suggestion, we have analyzed the expression pattern of some known miR-27a targets such as Plk2 and Pink1 as controls in the lung tissues of mice treated with miR-27a antagomir to ensure the efficacy of antagomir (Tian et al., 2014;  Kim et al., 2016). The results showed that all of these genes' expression are up-regulated in the lung tissues of miR-27a antagomir-treated mice, suggesting that the observed effects of miR-27a antagomir are most likely to be "on-target" effects. We have added this part into the main text (Supplementary Fig. 5e).

7)
In the TEM analysis of Cac3 m/m macrophages (lines 237), are these bacteria unincorporated into autophagosomes? Is there a difference observable between these and the proportion of Mtb +ve autophagosomes in the TEM from wild-type mice?
In our experiments, WT and Cac3 PB/PB macrophages were infected with H37Rv for 24 hours and then sent to TEM analysis. At this time-point, most of the H37Rv bacteria were not likely in autophagosomes, but present in lysosome or cytoplasm (Rahman et  al., 2014).

8) Figure 4i -Mtb infection perturbs calcium levels within seconds, as shown, but effects on autophagy and viability are on the time scale of hours. What is the effect on calcium levels at such later time points where evidence of autophagy regulation can actually be obtained?
Generally, Ca2 + signaling is a short-term signaling which is activated within seconds. However, Ca2 + ions usually functions through their binding to calmodulins, which in turn activate thes downstream effectors, thus maintaining the signaling for hours. (Berg et al., 2015) 9) Figure 5. The pathology of lungs in the infection model is presented largely without any quantification. Are these changes quantitatively significant between the genotypes? As per the reviewer's suggestion, we have provided the quantification of histopathology results as shown in Supplementary Fig. 4e. The data showed significant differences of the histopathological impairments between genotypes.

Minor points 1, Define PBMCs first time acronym is used (line 94)
As per the reviewer's suggestion, we have defined PBMCs as "peripheral blood mononuclear cell" in the main text. (Figure 1a-c)? An explanatory note to this effect would be good. As miR-27a's functional role in the regulation of Mtb infection remains uncharacterized, we choose it for our further study. As per the reviewer's suggestion, we have added an explanatory note in the revised manuscript.

3, Lines 111-112. Use of these chemical inhibitors does not conclusively demonstrate a TLR2/ERK/NfkappaB signaling pathway. This should be acknowledged in text.
In our study, selective inhibition of ERK MAPK signaling pathway by PD98059 or NF-κB signaling pathway by PDTC significantly suppressed Mtb-induced miR-27a's expression (Supplementary Fig. 1f), suggesting that Mtb infection may induce miR-27a expression via the activation of ERK MAPK signaling pathways and NF-κB signaling pathways. As per the reviewer's suggestion, we have edited this part accordingly in the revised manuscript.

4, Lines 118-124. For clarity, the authors should be explicit about how they conclude the initial infection/phagocytosis of Mtb is unaffected but the subsequent survival is affected (i.e. analysis of different time points within the data).
Thank the reviewer for instruction. For our CFU assay, we have measured the CFU at 2 hours to assure the uptake of Mtb is equal (Yang et al., 2016). The data showed that treatment of miR-27a mimic or inhibitor has no significant effect on the phagocytosis of Mtb in macrophages. (Fig. 1d, e and Supplementary Fig. 2a)

5, Line 135 -"influx" is not correct, "flux" is the correct term.
As per the reviewer's suggestion, we have changed the "influx" to "flux" in our revised manuscript.

6, Please check that panels 4a and 4b are in the correct order and referenced so in the text and Figure legends.
Thank the reviewer for correction, we have re-ordered these two panels in the revised manuscript (Fig. 4a, b). Figure 6. These may be mislabelled. Thank the reviewer for correction. We have re-labeled them in the revised manuscript.

Reviewer #3 (Remarks to the Author):
This manuscript describes the regulation of autophagy by Mycobacterium tuberculosis via microRNA (miRNA) miR-27a and an endoplasmic reticulum calcium transporter. This is a novel finding providing new insight to the intracellular survival of the tubercule bacillus. The work is exciting because M. tuberculosis encodes a number of proteins known to bind calcium. The authors provide a compelling story that convincingly shows this novel regulatory pathway. I found the work to be comprehensive and very well done. I recommend the acceptance of this paper. We thank the reviewer for appreciating the novelty and quality of our research work.

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