Mycobacterium tuberculosis response to cholesterol is integrated with environmental pH and potassium levels via a lipid metabolism regulator

Successful colonization of the host requires Mycobacterium tuberculosis (Mtb) to sense and respond coordinately to disparate environmental cues during infection and adapt its physiology. However, how Mtb response to environmental cues and the availability of key carbon sources may be integrated is poorly understood. Here, by exploiting a reporter-based genetic screen, we have unexpectedly found that overexpression of transcription factors involved in Mtb lipid metabolism altered the dampening effect of low environmental potassium concentrations ([K+]) on the pH response of Mtb. Cholesterol is a major carbon source for Mtb during infection, and transcriptional analyses revealed that Mtb response to acidic pH was augmented in the presence of cholesterol and vice versa. Strikingly, deletion of the putative lipid regulator mce3R had little effect on Mtb transcriptional response to acidic pH or cholesterol individually, but resulted specifically in loss of cholesterol response augmentation in the simultaneous presence of acidic pH. Similarly, while mce3R deletion had little effect on Mtb response to low environmental [K+] alone, augmentation of the low [K+] response by the simultaneous presence of cholesterol was lost in the mutant. Finally, a mce3R deletion mutant was attenuated for growth in foamy macrophages and for colonization in a murine infection model that recapitulates caseous necrotic lesions and the presence of foamy macrophages. These findings reveal the critical coordination between Mtb response to environmental cues and cholesterol, a vital carbon source, and establishes Mce3R as a transcription factor that crucially serves to integrate these signals.


Introduction
Bacterial colonization of its host requires adaptation of the bacteria to their local environment, which includes ionic signals such as protons (H + , pH levels), chloride (Cl -), and potassium (K + ) [1][2][3].Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, a disease that continues to be a leading cause of death from infectious diseases worldwide [4].The importance of appropriate sensing and response to the local environment for Mtb is underlined by the substantial attenuation in colonization ability of a mutant deleted for phoPR [5,6], a two-component system essential in the response of Mtb to acidic pH that also plays a significant role in its response to Cl - [1,2].Indeed, the attenuation of a ΔphoPR mutant for host colonization is so marked that it has been pursued as a possible vaccine candidate [7,8].Environmental cues further change in time and space during infection, and we have previously shown that spatial variations in local pH and Cl -levels exist even within a single lesion, and correlate to Mtb replication status [9].
Critically, bacteria are exposed during infection not just to a single signal at a time, but concurrently to multiple cues in their local environment.In the case of Mtb, we have shown that the bacteria have a synergistic transcriptional response to the simultaneous presence of acidic pH and high Cl -concentration ([Cl -]), two signals experienced concurrently within a maturing macrophage phagosome [2].There is further a relationship between the K + homeostasis of Mtb and its response to acidic pH/high [Cl -], with deletion of the CeoBC K + uptake system impairing the response of Mtb to these two environmental cues and negatively affecting the ability of Mtb to colonize its host [3].Besides PhoPR regulating Mtb response to both acidic pH and high [Cl -] [2], other examples of coordinated regulation in signal response are exemplified by additional key two-component systems such as DosRS(T) (nitric oxide stress and hypoxia) and PrrAB (acidic pH, high [Cl -], nitric oxide stress, hypoxia) [10,11].Of note, PrrA is essential in Mtb [11,12], while mutants in DosRS(T) show significant attenuation in several animal models of Mtb infection [13][14][15].These data emphasize the existence of interconnections between environmental signals, and the importance of coordination in response to these signals for successful host colonization by Mtb.
Alongside environmental stimuli, the availability of nutrients also plays a crucial role in bacterial adaptation to its host.Particularly for Mtb, cholesterol has been shown to be a major carbon source during infection, with Mtb mutants deficient in their ability to utilize cholesterol attenuated for host colonization [16,17].Strikingly, Mtb exhibits improved growth in acidic pH media when cholesterol or fatty acids are provided as carbon sources [18,19].Further, the transcriptional response of Mtb to Cl -was increased in the simultaneous presence of cholesterol [20].In addition, two studies have found that iron limitation increases expression of the cholesterol utilization genes in Mtb [21,22].Together, these data suggest the presence of intrinsic links between Mtb response to environmental (ionic) cues and its metabolism.However, the nature of these relationships and the regulatory factors that underlie them remain largely unexplored and unknown.
Here, building on our previous finding that disruption of Mtb K + homeostasis decreases the bacterial response to acidic pH and high [Cl -] [3], we first discover that limiting environmental K + levels also dampen Mtb response to acidic pH.Exploitation of a pH reporter Mtbbased transcription factor overexpression screen then unexpectedly revealed a connection between Mtb lipid metabolism regulation with control of the bacterial pH response in the presence of limiting [K + ].Focusing on the putative lipid metabolism regulator Mce3R, overexpression of mce3R partially rescued, while mce3R deletion conversely further repressed, the acidic pH response of Mtb in the presence of low external [K + ].The presence of cholesterol was found to increase Mtb transcriptional response to low [K + ], with global transcriptional analyses demonstrating that Mtb response to acidic pH was also augmented in the simultaneous presence of cholesterol and vice versa.Strikingly, deletion of mce3R resulted in loss of augmentation of the cholesterol response in the presence of acidic pH, while having minimal effect on Mtb transcriptional response to cholesterol or acidic pH alone.Similarly, augmentation of the low [K + ] response by the simultaneous presence of cholesterol was lost in the Δmce3R Mtb mutant.Lastly, we show that deletion of mce3R had significant consequences for Mtb host colonization, with the Δmce3R mutant attenuated for growth in foamy macrophages and colonization of C3HeB/FeJ mice during a six-week infection.Together, our findings reveal the intrinsic interconnections between Mtb response to acidic pH, low [K + ], and cholesterol, and establish Mce3R as a critical mechanism through which integration of these signals is accomplished in Mtb.

Environmental K + levels modulate Mtb transcriptional response to acidic pH
To investigate the impact of environmental potassium (K + ) levels on the response of Mtb to acidic pH, we exposed our previously established pH-responsive Mtb reporter, rv2390c'::GFP [2], to standard 7H9, pH 7 medium, or 7H9, pH 5.85 medium with sufficient K + (7.35 mM, the concentration present in standard 7H9 medium) or a K + concentration limited to 0.1 mM.As shown in Fig 1A, K + limitation strikingly decreased the induction of rv2390c'::GFP fluorescence signal observed at acidic pH.While limiting [K + ] to 0.1 mM in pH 5.85 media slowed the initial rate of Mtb growth, there was no significant difference in optical density reached at the later time points of the assay (day 9 onwards) (Fig 1B).The observed decrease in rv2390c':: GFP induction in the 0.1 mM [K + ], pH 5.85 condition was thus not simply a result of reduced bacterial growth.This decrease in acidic pH response was further [K + ]-dependent, with increased dampening of reporter induction with decreasing [K + ] (Fig 1C).Importantly, this effect of K + limitation on the acidic pH response of Mtb was unidirectional, as the bacterial response to low [K + ], tested using the K + reporter Mtb strain kdpF'::GFP [3], was not influenced by changes in environmental pH (Fig 1D).
Together, these assays reveal a previously unknown impact of environmental [K + ] levels on the response of Mtb to acidic pH.

Lipid metabolism regulators modulate the interplay between environmental [K + ] and Mtb pH response
To investigate the regulatory mechanism(s) underlying the relationship between environmental [K + ] and the acidic pH response of Mtb, a screening approach was employed using an arrayed inducible transcription factor overexpression library generated in the background of a pH-responsive rv2390c'::luciferase reporter [11].7H9, pH 7, 0.05 mM K + or 7H9, pH 5.85, 1.6 mM K + media served as negative and positive control conditions respectively, with an empty vector control included.1.6 mM K + was empirically selected for use in the positive control condition, given rv2390c'::luciferase induction at pH 5.85 was maximal at that K + concentration, and similar to that observed in standard 7H9 medium (S1 Fig) .Two K + limiting concentrations, 0.1 and 0.05 mM, both at pH 5.85, were used as the test conditions.The fold induction of rv2390c'::luciferase reporter signal in each test condition compared to the negative control was calculated as relative light units (RLU)/OD 600 to account for any variations in bacterial growth.The arrayed set-up of the screen removed any concerns of bottleneck effects and enabled immediate identification of hits of interest.
Unexpectedly, a key class of hits identified from the screen were regulators involved in lipid (cholesterol or fatty acid) metabolism (Fig 2A and S1 Table).In particular, overexpression of rv1219c and mce3R partially rescued the dampening of rv2390c'::luciferase signal at pH 5.85 with limiting [K + ] (Fig 2A and S1 Table).Conversely, overexpression of mce1R, kstR, mce2R, rv1129c, and rv1816 repressed rv2390c'::luciferase induction even more compared to the empty vector control (Fig 2A and S1 Table).These phenotypes were validated by comparing uninduced (ethanol, EtOH) conditions versus induced (200 ng/ml anhydrotetracycline, ATC) conditions for each transcription factor overexpression strain, with outcomes consistent with the screen results (comparison to empty vector) obtained in all cases (Fig 2B).As noted above, these seven transcription factors are known or predicted to be involved in lipid metabolism.Specifically, mce1R, mce2R, rv1219c, and rv1816 have previously been shown to be involved in fatty acid metabolism [23][24][25][26][27], kstR is a key regulator of cholesterol β-oxidation [28,29], and rv1129c is involved in propionyl-CoA metabolism [30].mce3R is predicted to be involved in lipid metabolism due to the homology of the Mce3 complex that it regulates to the other Mce complexes [31][32][33][34], and the role of genes such as fadE17 and fadE18 that lie within the Mce3R regulon [35].
We decided to pursue here further study of Mce3R, given its still poorly understood role in Mtb biology.A deletion mutant strain of mce3R was generated and tested in the same experimental conditions as in the original rv2390c'::luciferase transcription factor overexpression screen.As shown in Fig 2C, in contrast to overexpression of mce3R which partially rescued the Mtb pH response phenotype in the presence of low [K + ], deletion of mce3R resulted in the converse phenotype, with a significant reduction in pH regulon gene expression in the mutant strain in acidic pH + K + -limiting conditions, compared to the parental wild type (WT) strain.This was observed not just with rv2390c, but also with both the two other pH-responsive genes tested, with complementation of the Δmce3R strain restoring gene induction to WT levels in all cases (Fig 2C ).
Taken together, these results indicate that factors related to lipid metabolism regulation play a role in modulating the interplay between external K + levels and the response of Mtb to the critical environmental cue of acidic pH.

Mtb transcriptional response to acidic pH, low [K + ], and cholesterol are linked
Given the intriguing connection revealed by the screen between Mtb lipid metabolism regulation, acidic pH response, and environmental [K + ], we next pursued investigation of how these three facets might be related at the transcriptional level.Examination of the impact of K + limitation on the transcriptional response of Mtb to cholesterol showed that it also dampened induction of the five tested Mtb cholesterol regulon genes (Fig 3A).Interestingly, we also observed a reverse association, where cholesterol further induced the expression of genes in the K + regulon, in the presence of K + -limiting conditions (Fig 3B).These data relate the presence of cholesterol to environmental K + , further supporting a complex interplay between lipid metabolism, environmental K + levels, and Mtb response to acidic pH.
Given the extensive transcriptional changes known to result from Mtb exposure to acidic pH or cholesterol individually [1,17,36], we next sought to obtain a comprehensive  (A and B) Lipid metabolism regulator hits from reporter-based, inducible transcription factor (TF) overexpression screen.A library of inducible TF overexpression plasmids (P 1 '::TF-FLAG-tetON) in the background of a Mtb(rv2390c'::luciferase) strain was screened for their response to acidic pH in the presence of low [K + ].TF overexpression was induced by adding 200 ng/ml of anhydrotetracycline (ATC) 1 day before Mtb was exposed to K + -free 7H9, pH 7 medium supplemented with 0.05 mM K + , or K + -free 7H9, pH 5.85 media supplemented with 1.6 mM, 0.1 mM, or 0.05 mM K + .9 days postexposure (continuous ATC presence), light output (relative light units, RLU) and OD 600 were measured.Fold induction compares RLU/OD 600 in each condition to RLU/OD 600 in the control 0.05 mM K + 7H9, pH 7 condition.(A) shows results of the lipid metabolism regulator hits, together with an empty vector plasmid control.(B) shows validation of the screen hits in (A), with each hit TF compared to its uninduced control (ethanol, "EtOH", as a carrier control).In Complementarily, the RNAseq data showed that the presence of cholesterol also affected differential gene expression of Mtb in response to acidic pH (Fig 4D and S3 Table).In this case, the presence of cholesterol increased expression of a subset of pH regulon genes, with expression of 118 differentially expressed genes increased (genes differentially expressed log 2fold change �1 in the pH 5.7 condition; log 2 -fold change �0.6 between cholesterol, pH 5.7 and 7H9, pH 5.7 conditions; p<0.05,FDR<0.01 in both sets), but also interestingly reduced expression of a different subset of genes, with 67 differentially expressed genes decreased (genes differentially expressed log 2 -fold change �1 in the pH 5. alone downregulates expression of these genes.This finding indicates an overlap in a subset of the cholesterol and pH regulons in Mtb; expression of these genes are regulated in opposite directions by the two signals when present individually (induced by acidic pH, repressed by cholesterol), with cholesterol appearing to be the dominant signal when Mtb is exposed to both cues concurrently.
Focusing on genes in the acidic pH regulon that are not also differentially expressed in the presence of cholesterol alone, qRT-PCR analysis showed a concentration dependence in the augmentation of gene expression induction at acidic pH with increasing cholesterol concentration (Fig 4E).pH level-dependence of induction of the acidic pH regulon genes tested was also observed in both standard 7H9 and cholesterol conditions (Fig 4F).Overall, these findings show the global cross-regulation between cholesterol and pH response in Mtb, with the concurrent presence of each factor strongly influencing the expression of genes in each regulon.

Mce3R regulates Mtb response to cholesterol only in the context of acidic pH
Our results above raise the question of how Mtb response to acidic pH and cholesterol is cross-regulated.To gain insight into possible regulatory mechanisms underlying the synergy in Mtb transcriptional response to cholesterol and acidic pH in the concurrent presence of both cues, we first tested the effect of a phoPR deletion on these responses.PhoPR is a wellestablished two-component system known to play a crucial role in Mtb response to the linked environmental cues of acidic pH and Cl - [1,2].Consistent with this, we observed that a ΔphoPR mutant exhibited significantly reduced induction of acidic pH regulon genes in both standard 7H9 or cholesterol media as a base (S2A The putative lipid metabolism regulator Mce3R had been found as a robust hit in the transcription factor overexpression library screen described above, which first revealed the ability of lipid metabolism regulators to modulate the interplay between environmental K + levels and the response of Mtb to acidic pH.While Mce3R is known to regulate the mce3 operon and associated upstream genes [31][32][33], its role in the overall response of Mtb to cholesterol remains unclear.To determine if Mce3R might also play a role in coordinating Mtb acidic pH and cholesterol responses, we performed global RNAseq analysis on the Δmce3R mutant and compared the transcriptional profiles obtained to those of WT Mtb.The same four conditions utilized above for WT Mtb were used here, namely: (i) 7H9, pH 7 (control), (ii) 7H9, pH 5.7, (iii) cholesterol, pH 7, and (iv) cholesterol, pH 5.7 media.A first observation was that mce3R deletion had little effect on global transcriptional response of Mtb to acidic pH or cholesterol when either condition was present alone (Fig 5A and 5B and S4 and S5 Tables).Strikingly however, in the dual cholesterol, pH 5.7 condition, a subset of genes associated with the cholesterol regulon were significantly less induced in the Δmce3R Mtb mutant compared to WT (Fig 5C and S6 Table).Follow-up qRT-PCR experiments validated that mce3R deletion only affected expression of cholesterol regulon genes in Mtb in the context of the simultaneous presence of cholesterol and acidic pH, with complementation restoring gene expression to WT levels (Fig 5D and 5E).Markedly, for the Δmce3R mutant, the induction levels of the cholesterol regulon genes in the dual cholesterol, pH 5.  Given (i) the results above, (ii) our finding that cholesterol also augments Mtb response to low [K + ] (Fig 3B ), and (iii) the original identification from our screen of Mce3R as a regulator able to modulate the interplay between environmental K + levels and the response of Mtb to acidic pH, we next sought to test if Mce3R would also have a role in coordinating Mtb response in the simultaneous presence of both cholesterol and low [K + ].WT, Δmce3R, and the complemented Δmce3R Mtb mutant (mce3R*) were exposed to: (i) K + -free 7H9, pH 7, or (ii) K + -free cholesterol, pH 7 media, for 4 hours before RNA were extracted for gene expression analysis.Strikingly, the ability of cholesterol to augment induction of the K + regulon genes was largely lost in the Δmce3R mutant, even as deletion of mce3R had no effect on induction of the K + regulon genes in the absence of cholesterol (Fig 6A).The dampening of cholesterol gene Together, these results excitingly identify Mce3R as a transcription factor that specifically acts to coordinate Mtb response in the presence of at least two environmental signals (cholesterol + acidic pH, low [K + ] + cholesterol, acidic pH + low [K + ]), with little effect when only one signal is present.There is further directionality in its activity, as in each case, mce3R deletion affected response to only one of the two signals concurrently present.These findings emphasize the interconnectedness between Mtb response to acidic pH, low [K + ], and cholesterol, and the multifaceted role of Mce3R in the coordinated regulation of Mtb response to its local environment.infection.While Mtb actively prevents complete maturation of the phagosome, it is still exposed to slightly decreased pH levels during macrophage infection [39].These results thus support a role of Mce3R in the ability of Mtb to respond appropriately to its local environment for continued growth.

A Δmce3R Mtb mutant is attenuated for host colonization in the presence of lipids
Finally, we examined the role of Mce3R in Mtb infection of a whole animal host using the C3HeB/FeJ murine model.Importantly, this mouse strain recapitulates key lesion types observed during human infection [40][41][42].Of pertinence here, foamy macrophages are present at 6 weeks post-infection, both in macrophage-rich lesions and in the cuff surrounding caseous necrotic lesions that are the hallmark of tuberculosis disease (Fig 7B) [43].The mice were infected with WT, Δmce3R, or mce3R* Mtb, and the infection allowed to progress for 2 or 6 weeks.We did not observe significant differences in bacterial load among the three strains at the 2-week time point (Fig 7C

Discussion
Throughout the course of infection, Mtb encounters a complex and dynamic environmental milieu that plays a crucial role in shaping its interaction with its host.This milieu encompasses signals originating from the cellular and tissue microenvironment, which often reflect the host's immune response, and changes to available nutrient sources.There have been extensive studies seeking to understand the response of Mtb to specific environmental signals and nutrients, and the regulation underlying such responses.In contrast, significantly less is known regarding the interplay between Mtb response to particular environmental stimuli and available nutrients, and the regulatory mechanisms that enable the integration of such responses.Our study here reveals the intrinsic relationships between Mtb response to low [K + ], acidic pH, and cholesterol, with synergy in the acidic pH and cholesterol response, and dampening of Mtb response to both signals in the presence of low [K + ] (Fig 8A).Concurrent exposure to acidic pH and cholesterol is expected to be a critical aspect of the local environment during Mtb infection, in the context of bacterial residence in phagosomes of foamy macrophages, a major host cell type observed not just in animal models of infection, but also in human infections [40,41,44,45].Indeed, Mtb growth in acidic pH media is improved when cholesterol is provided as the carbon source [18], and our finding of synergy in Mtb transcriptional response to these two signals provides a crucial new dimension to the understanding of Mtb adaptive biology in this environment.
Changes in expression of bacterial "virulence factors", driven by changes in the presence of specific metabolites, have been increasingly described for different bacterial pathogens [46].This includes increased expression of leucocidins in Staphylococcus aureus in the presence of pyruvate [47], and induced expression of Salmonella pathogenicity island 2 genes in the presence of succinate [48].Our results demonstrating the reciprocal augmentation of Mtb response to acidic pH and cholesterol in the concurrent presence of both signals, and the increased expression of K + -responsive genes in the simultaneous presence of cholesterol, expands on this facet of bacterial biology.In particular, together with previous studies that have shown increased transcription of genes in the Cl -regulon in the simultaneous presence of cholesterol [20], and increased expression of cholesterol utilization genes in iron-limiting conditions [21,22], our findings support the existence of an intrinsic interplay between Mtb response to environmental cues and the critical nutrient of cholesterol.
Strikingly, we identify Mce3R as a transcription factor that critically functions in the integration of these signals.Specifically, a Δmce3R mutant fails to synergistically upregulate genes in the cholesterol regulon in the simultaneous presence of acidic pH, and in the K + regulon in the simultaneous presence of cholesterol, even while response to each signal individually is unaffected (  [25,49].Whether and how Mce3R binding to DNA may be affected by cholesterol, and whether interacting partners exist that aid in transducing the external stimuli to Mce3R, are thus areas for future investigation.Of interest, from the RNAseq data, induction of the transcription factor rv1219c and its regulon (rv1216c -rv1218c) were specifically downregulated in the cholesterol/acidic pH dual condition, but not in acidic pH or cholesterol alone, upon mce3R deletion.Rv1219c is a transcription factor that acts as an autorepressor, repressing expression of the ABC transporter system encoded by rv1217c-rv1218c that reside in the same operon, and has been studied primarily in the context of drug resistance [27,50,51].Intriguingly, long chain fatty acid CoA derivatives have been reported to bind in a large cavity in the C-terminal ligand-binding domain of Rv1219c, affecting its ability to bind target DNA [27].Whether cholesterol may also bind to this ligand-binding domain of Rv1219c, and how Mce3R may work with Rv1219c in coordinating Mtb cholesterol metabolism with environmental pH, are thus also exciting questions for follow-up studies.Finally, it was recently reported that a Δmce3R Mtb mutant showed qualitative differences in the amount of some membrane lipids, and was less susceptible to oxidative stress [52].Further studies are required to determine how changes in membrane lipid profile may affect signal transduction, and whether Mce3R may also act in integrating oxidative stress signals with cholesterol response in Mtb.
In accord with our finding that a transcriptional effect of mce3R deletion on Mtb response to cholesterol was only revealed in the context of acidic pH, the Δmce3R mutant showed a reduced bacterial load in the C3HeB/FeJ murine infection model only at the later 6 week time point, when Mtb can be found in foamy macrophages.Particularly given the distinct environments that Mtb is exposed to spatially during infection (e.g.residence in foamy macrophages versus extracellularly in the necrotic core of a caseous lesion) [9], it will be intriguing in follow-on studies to determine if Mce3R function is differentially important for Mtb survival and growth depending on sublocation within the lung/lesion.In contrast to our findings, we note that a recent report in a guinea pig model of Mtb infection indicated increased loads of Δmce3R Mtb as compared to WT Mtb in the lungs at 4 and 8 weeks postinfection [52].This phenotype could not, however, be complemented, with the complemented strain showing slightly higher levels in bacterial load than the mce3R deletion mutant [52].It is thus possible that the differences observed in that study are due to other mutations inadvertently present in the Δmce3R strain used, or reflective of the different Mtb strain and infection model utilized.
As in mammalian cells, K + is also the most abundant cation in bacterial cells, and the importance of K + in host colonization is becoming increasingly appreciated [3,53,54].Our unexpected finding linking Mtb lipid metabolism regulators to the bacterium's K + response opens a new facet of study in understanding how K + response/homeostasis integrates with the metabolism of a bacterium.Distinct [K + ] gradients exist within the mammalian host, with [K + ] high intracellularly, but low extracellularly [55,56], and multiple K + channels/ transporters exist through which tight control of local [K + ] is maintained [54,57,58].While the exact environmental [K + ] that Mtb may be exposed to in a host is unknown, it is likely to encounter changing K + levels during the course of infection, both as it progresses from the extracellular to the intracellular environment upon first engulfment by a phagocyte, and in the context of residence intracellularly versus extracellularly in the caseous necrotic lesion core.Intriguingly, cholesterol regulation of K + channel activity has been demonstrated in mammalian systems, with the inwardly rectifying K + (Kir) channels shown to possess cholesterol-binding sites [59,60].Regulation of the prokaryotic homolog of Kir (KirBac1.1)by cholesterol has also been demonstrated [61].Cholesterol binding usually downregulates Kir channel function, although it has been shown to enhance activity of certain Kir channels, such as Kir3.4 [62].In Mtb, the CeoBC K + uptake system is critical in maintaining K + homeostasis [3,63], with a ΔceoBC mutant diminished in its ability to respond to acidic pH and high [Cl -], and attenuated for host colonization [3].Whether cholesterol directly regulates CeoBC activity in Mtb, and more broadly K + uptake systems in other pathogens, is an interesting question for future study.
Exposure to different environmental signals with a simultaneous change in available nutritional source is a widespread phenomenon for bacterial pathogens and not unique to Mtb.We propose that further study of bacterial responses to concurrent environmental and nutritional cues will yield important new insight into bacterial signal integration and their underlying mechanisms.Unique nodes represented by regulatory factors that specifically act to coordinate bacterial environmental response to multiple signals and thus adaptation, such as Mce3R as identified here, further represent exciting new candidates for therapeutic targeting [64].

Ethics statement
All animal protocols in this research followed the guidelines from The National Institutes of Health "Guide for Care and Use of Laboratory Animals".All animal protocols (#B2021-139) were reviewed and approved by the Institutional Animal Care and Use Committee at Tufts University, in accordance with guidelines from the Association for Assessment and Accreditation of Laboratory Animal Care, the US Department of Agriculture, and the US Public Health Service.

Mtb strains and culture
Mtb cultures were propagated and maintained as previously described [65]."Standard 7H9 medium" refers to 7H9 broth supplemented with 10% OADC, 2% glycerol, and 0.05% Tween 80, with 100 mM MOPS used for buffering to pH 7.0, or 100 mM MES used for buffering to acidic pH values [65].K + -free and cholesterol media were prepared as previously described [3,20].In particular, unless otherwise specified for experiments where cholesterol concentrations were altered, cholesterol media consisted of 7H9 broth (base powder only) supplemented with 0.5 g/l fatty acid-free bovine serum albumin, 14.5 mM NaCl, 0.2 mM cholesterol, and 0.1% tyloxapol [20].Buffering was with 100 mM MOPS for pH 7.0 medium, or 100 mM MES for acidic pH media.All antibiotics were added as appropriate at the following concentrations: 100 μg/ml streptomycin, 50 μg/ml hygromycin, 50 μg/ml apramycin, and 25 μg/ml kanamycin.Strains for in vitro assays were in the CDC1551 background, while infection assays were carried out with strains in the Erdman background.The rv2390c'::GFP, kdpF'::GFP, and smyc':: mCherry reporters, and the ΔphoPR strain and its complement have all been previously described [2,3].The Δmce3R mutant and its complement were constructed as previously described [2], with the Δmce3R mutation consisting of a deletion beginning at the mce3R start codon (as annotated in the Erdman Mtb strain) through 26 bp from the mce3R stop codon.The rv2390c'::luciferase transcription factor overexpression library has been previously described [11].

Reporter Mtb strain broth assays and Mtb growth assays
Reporter strains at log-phase were subcultured to an OD 600 = 0.05 and resuspended in the appropriate media in standing T25 flasks with filter caps.These were: (i) 7H9, pH 7, (ii) 7H9, pH 5.85, and (iii) K + -free 7H9 supplemented with 0.1 mM K + , pH 5.85, for assays with the rv2390c'::GFP reporter.For assays with the kdpF'::GFP reporter, media conditions were: (i) 7H9, pH 7, (ii) 7H9, pH 5.7, (iii) K + -free 7H9, pH 7, and (iv) K + -free 7H9, pH 5.7.For broth assays conducted in K + -free media, an additional wash step with K + -free 7H9, pH 7 medium was performed prior to resuspending the cultures in the final assay media.At each time point, aliquots of the cultures were taken and fixed in 4% paraformaldehyde (PFA) in phosphatebuffered saline (PBS).Fixed samples were subsequently pelleted and resuspended in PBS + 0.1% Tween 80 for flow cytometry analysis.To disrupt any clumps, each sample was passaged 6x through a tuberculin syringe with a 25G x 5/8" needle just prior to running on the flow cytometer.Reporter GFP signal was measured using a BD FACSCalibur flow cytometer, with GFP signal/bacterium from 10,000 Mtb cells recorded for each experimental run (three independent experimental runs performed).Mean GFP fluorescence values for each experimental run were obtained by analyzing the data using FlowJo software (BD).
For growth assays, log-phase Mtb was used to inoculate cultures at a starting OD 600 = 0.05 in standing T25 flasks with filter caps, and OD 600 measured at indicated time points.Media conditions used were (i) 7H9, pH 7, (ii) 7H9, pH 5.7, (iii) cholesterol media, pH 7, (iv) cholesterol media, pH 5.7, (v) K + -free 7H9, pH 7, supplemented with 0.05 mM KCl, and (vi) K + -free cholesterol media, pH 7, supplemented with 0.05 mM KCl.As above, for growth assays conducted in K + -free media as base, an additional wash step with K + -free 7H9, pH 7 medium was performed prior to resuspending the cultures in the final assay media.

rv2390c'::luciferase transcription factor overexpression library screen
To screen the rv2390c'::luciferase, tetracycline-inducible transcription factor overexpression (TFOE) library, thawed TFOE Mtb strains in 96-well plates (35 μl volume) were mixed with 165 μl of 7H9, pH 7 medium supplemented with 25 μg/ml kanamycin and 50 μg/ml hygromycin.The plates were then incubated for 11 days at 37˚C in a 5% CO 2 incubator.Subsequently, the TFOE rv2390c'::luciferase strains were subcultured at a 1:10 dilution into 100 μl of fresh 7H9, pH 7 medium with appropriate antibiotics and incubated for an additional eight days.After eight days of growth, 200 ng/ml anhydrotetracycline (ATC) was added to induce overexpression of each transcription factor for 24 hours.Following the induction, all samples were transferred to a 96-well v-bottom plate (Corning Costar) and pelleted at 3,000 rpm for 10 minutes.The samples were then washed once with K + -free 7H9, pH 7. Each strain was then inoculated at a 1:10 dilution into 100 μl of the following media conditions: (i) K + -free 7H9, pH 7, supplemented with 0.05 mM KCl, (ii) K + -free 7H9, pH 5.85, supplemented with 1.6 mM KCl, (iii) K + -free 7H9, pH 5.85, supplemented with 0.1 mM KCl, and (iv) K + -free 7H9, pH 5.85, supplemented with 0.05 mM KCl, in clear-bottom white 96-well plates (Corning Costar).The cultures were then incubated for nine days at 37˚C in a 5% CO 2 incubator.All media contained 50 μg/ml hygromycin and 25 μg/ml kanamycin, as well as 200 ng/ml ATC for continued transcription factor overexpression.Following this nine-day incubation, luminescence was assessed using the Bright-Glo luciferase assay system (Promega) as previously described, with both the light output (relative light units, RLU), and the OD 600 measured using a Biotek Synergy Neo2 multi-mode microplate reader [11].Fold induction was determined by comparing the RLU/OD 600 values of the samples from each environmental condition to the RLU/OD 600 value of Mtb in the K + -free 7H9, pH 7, 0.05 mM KCl control condition.

RNA sequencing and qRT-PCR analyses
For RNA sequencing (RNAseq) and qRT-PCR analyses, log-phase Mtb cultures (OD 600 ~0.6) were used to inoculate standing T25 flasks with filter caps at an OD 600 = 0.3, containing 10 ml of the following media: (i) 7H9, pH 7.0, (ii) 7H9, pH 5.7, (iii) cholesterol, pH 7.0, or (iv) cholesterol, pH 5.7.For transcriptional analysis of Mtb under K + -free conditions, an additional wash step with K + -free 7H9, pH 7, was performed prior to inoculation of the culture into the various test conditions: (i) K + -free 7H9, pH 7, supplemented with 0.05 mM KCl (control), (ii) K + -free 7H9, pH 5.85, supplemented with 1.6 mM KCl, (iii) K + -free 7H9, pH 5.85, supplemented with 0.1 mM KCl, or (iv) K + -free 7H9, pH 5.85, supplemented with 0.05 mM KCl.After four hours of exposure to the specific environmental conditions, Mtb samples were collected and RNA isolated as previously described [1].For RNAseq, two biological replicates were prepared for each condition.Library preparation was performed by the Tufts University Genomics Core Facility using the Illumina stranded total RNA with Ribo-Zero Plus kit, and barcoded samples were pooled and sequenced on an Illumina HiSeq 2500 (single-end 100 bp reads).RNAseq data were analyzed using the Geneious program (version 2023.0.4).Specifically, raw data were trimmed using BBDuk with the following parameters: trim adapters (All Truseq), Kmer lengh 27, trim low quality on both ends with minimum quality 30, discard short reads with minimum length 36 bp, minimum entropy 0.1, entropy window size 4 and entropy Kmer size 5.
Trimmed sequences were assembled onto the CDC1551 genome (GenBank AE000516.2) using the Bowtie2 mapper [66] with end-to-end alignment.Gene expression levels were then calculated and excluded ambiguously mapped reads from calculation, with comparison of expression levels performed via DEseq2 [67].A log 2 -fold change �1 (= fold change �2) was used as the cutoff when first setting the list of genes differentially expressed in a given environmental condition/in WT Mtb.A slightly less stringent cutoff of log 2 -fold change �0.6 (= fold change �1.5) was then used when comparing the effect of a second environmental condition on the first condition (e.g.effect of acidic pH on cholesterol regulon genes or vice versa) or between strains (i.e.Δmce3R versus WT), to enable capture of a more nuanced overview of these comparisons within a regulon.
qRT-PCR experiments were carried out and data analyzed as previously described [3].In brief, cDNA was generated from 250 ng of isolated RNA using the iScript cDNA synthesis kit (Bio-Rad).qRT-PCR was carried out using the iTaq universal SYBR Green supermix kit (Bio-Rad) on an Applied Biosystems StepOnePlus real time PCR system, with each run performed in triplicate.sigA was used as the control gene, and fold induction calculated using the ΔΔCT method [68].

Macrophage culture and infections
Bone marrow-derived macrophages were isolated from C57BL/6J wild type mice obtained from Jackson Laboratories.The cells were cultured in DMEM supplemented with 10% FBS, 15% L929-cell conditioned media, 2 mM L-glutamine, 1 mM sodium pyruvate, and penicillin/ streptomycin when needed, and maintained in a 37˚C incubator with 5% CO 2 .To induce the formation of foamy macrophages, macrophages were pre-treated with macrophage medium supplemented with oleate/albumin (0.42 mM sodium oleate, 0.35% BSA) complexes 24 hours prior to infection, following a previously established protocol [37,38].Macrophage infections with Mtb were carried out as previously described [2,65].For enumeration of colony forming units (CFUs), macrophages were lysed using a solution of water containing 0.01% sodium dodecyl sulfate, and serial dilutions plated on 7H10 agar plates supplemented with 100 μg/ml cycloheximide.

Mouse Mtb infections
C3HeB/FeJ wild type mice from Jackson Laboratories were intranasally infected with 10 3 CFUs of Mtb in a volume of 35 μl, while under light anesthesia with 2% isoflurane [9,11,20].Following sacrifice using CO 2 at 2 or 6 weeks post-infection, the left lobe and accessory right lobe of the lungs were homogenized in PBS containing 0.05% Tween 80.Serial dilutions of the homogenates were plated on 7H10 agar plates supplemented with 100 μg/ml cycloheximide for quantification of CFUs.

Quantification and statistical analysis
GraphPad Prism was used for all statistical analyses.Details of statistical tests performed are indicated in the figure legends.p<0.05 was considered significant.

Fig 1 .
Fig 1.Environmental K + levels modulate Mtb transcriptional response to acidic pH.(A and B) rv2390c'::GFP reporter response to acidic pH is dampened in the presence of low [K + ].Mtb(rv2390c'::GFP) was grown in 7H9, pH 7.0 or pH 5.85 media ([K + ] = 7.35 mM), or in K + -free 7H9, pH 5.85 medium, supplemented with 0.1 mM K + .Samples were taken at indicated time points and either fixed for analysis of GFP induction by flow cytometry (A) or OD 600 measured (B).Data are shown as means ± SEM from 3 experiments.p-values were obtained with an unpaired t-test with Welch's correction and Holm-Sidak multiple comparisons, and compare the 7H9, pH 5.85 to the 0.1 mM K + , pH 5.85 condition.N.S. no significant, ** p<0.01.(C) Dampening of Mtb response to acidic pH by low K + is concentration dependent.Mtb(rv2390c'::GFP) was grown in 7H9, pH 5.85 medium ("control"), or in K + -free 7H9, pH 5.85 medium, supplemented with indicated amounts of K + .Samples were taken 9 days post-assay start, fixed, and GFP induction analyzed by flow cytometry.Data are shown as means ± SEM from 3 experiments.(D) Mtb response to low [K + ] is not affected by environmental pH.Mtb(kdpF'::GFP) was grown in 7H9, pH 7.0 or pH 5.7, or in K + -free 7H9, pH 7 or pH 5.7 media.Samples were taken at indicated time points, fixed, and GFP induction analyzed by flow cytometry.Data are shown as means ± SEM from 3-4 experiments.For all flow cytometry assays, mean GFP fluorescence/Mtb was obtained from measurement of 10,000 Mtb cells in each experimental run.The numerical data underlying the graphs shown in this figure are provided in S1 Data.https://doi.org/10.1371/journal.pgen.1011143.g001

Fig 2 .
Fig 2. A rv2390c'::luciferase reporter transcription factor overexpression screen identifies lipid metabolism regulators as modulators of the interplay between environmental [K + ] and Mtb pH response.(A and B)Lipid metabolism regulator hits from reporter-based, inducible transcription factor (TF) overexpression screen.A library of inducible TF overexpression plasmids (P 1 '::TF-FLAG-tetON) in the background of a Mtb(rv2390c'::luciferase) strain was screened for their response to acidic pH in the presence of low [K + ].TF overexpression was induced by adding 200 ng/ml of anhydrotetracycline (ATC) 1 day before Mtb was exposed to K + -free 7H9, pH 7 medium supplemented with 0.05 mM K + , or K + -free 7H9, pH 5.85 media supplemented with 1.6 mM, 0.1 mM, or 0.05 mM K + .9 days postexposure (continuous ATC presence), light output (relative light units, RLU) and OD 600 were measured.Fold induction compares RLU/OD 600 in each condition to RLU/OD 600 in the control 0.05 mM K + 7H9, pH 7 condition.(A) shows results of the lipid metabolism regulator hits, together with an empty vector plasmid control.(B) shows validation of the screen hits in (A), with each hit TF compared to its uninduced control (ethanol, "EtOH", as a carrier control).In (B), data are shown as means ± SEM from three experiments, and p-values were obtained with an unpaired t-test with Welch's correction.N.S. not significant, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.(C) Mce3R modulates the interplay between environmental [K + ] and Mtb pH response.Log-phase WT, Δmce3R and mce3R* (complemented mutant) were exposed for 4 hours to K + -free 7H9, pH 7 medium supplemented with 0.05 mM K + or K + -free 7H9, pH 5.85 media supplemented with 1.6 mM or 0.05 mM K + , before RNA was extracted for qRT-PCR analysis.Fold change is as compared to the 0.05 mM K + 7H9, pH 7 condition.sigA was used as the control gene, and data are shown as means ± SEM from 3 experiments.p-values were obtained with an unpaired t-test with Welch's Fig 2. A rv2390c'::luciferase reporter transcription factor overexpression screen identifies lipid metabolism regulators as modulators of the interplay between environmental [K + ] and Mtb pH response.(A and B)Lipid metabolism regulator hits from reporter-based, inducible transcription factor (TF) overexpression screen.A library of inducible TF overexpression plasmids (P 1 '::TF-FLAG-tetON) in the background of a Mtb(rv2390c'::luciferase) strain was screened for their response to acidic pH in the presence of low [K + ].TF overexpression was induced by adding 200 ng/ml of anhydrotetracycline (ATC) 1 day before Mtb was exposed to K + -free 7H9, pH 7 medium supplemented with 0.05 mM K + , or K + -free 7H9, pH 5.85 media supplemented with 1.6 mM, 0.1 mM, or 0.05 mM K + .9 days postexposure (continuous ATC presence), light output (relative light units, RLU) and OD 600 were measured.Fold induction compares RLU/OD 600 in each condition to RLU/OD 600 in the control 0.05 mM K + 7H9, pH 7 condition.(A) shows results of the lipid metabolism regulator hits, together with an empty vector plasmid control.(B) shows validation of the screen hits in (A), with each hit TF compared to its uninduced control (ethanol, "EtOH", as a carrier control).In (B), data are shown as means ± SEM from three experiments, and p-values were obtained with an unpaired t-test with Welch's correction.N.S. not significant, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.(C) Mce3R modulates the interplay between environmental [K + ] and Mtb pH response.Log-phase WT, Δmce3R and mce3R* (complemented mutant) were exposed for 4 hours to K + -free 7H9, pH 7 medium supplemented with 0.05 mM K + or K + -free 7H9, pH 5.85 media supplemented with 1.6 mM or 0.05 mM K + , before RNA was extracted for qRT-PCR analysis.Fold change is as compared to the 0.05 mM K + 7H9, pH 7 condition.sigA was used as the control gene, and data are shown as means ± SEM from 3 experiments.p-values were obtained with an unpaired t-test with Welch's

Fig 3 .
Fig 3. Mtb transcriptional response to low [K + ] and cholesterol are linked.(A) Low [K + ] dampens Mtb response to cholesterol.Log-phase Mtb was exposed for 4 hours to (i) 7H9, pH 7, (ii) cholesterol, pH 7, or (iii) K + -free cholesterol, pH 7 media, before RNA was extracted for qRT-PCR analysis.[K + ] = 7.35 mM in both conditions (i) and (ii).(B) Cholesterol augments Mtb response to low [K + ].Log-phase Mtb was exposed for 4 hours to (i) 7H9, pH 7, (ii) K + -free 7H9, pH 7, or (iii) K + -free cholesterol, pH 7 media, before RNA was extracted for qRT-PCR analysis.Fold change is as compared to the 7H9, pH 7 condition in all cases.sigA was used as the control gene, and data are shown as means ± SEM from 3 experiments.p-values were obtained with an unpaired t-test with Welch's correction.*** p<0.001, **** p<0.0001.The numerical data underlying the graphs shown in this figure are provided in S1 Data.https://doi.org/10.1371/journal.pgen.1011143.g003

Fig 4 .
Fig 4. Mtb transcriptional response to acidic pH and cholesterol are linked globally.(A and D) Global changes in Mtb response to cholesterol and acidic pH in the simultaneous presence of both signals.Log-phase Mtb was exposed for 4 hours to (i) 7H9, pH 7, (ii) 7H9, pH 5.7, (iii) cholesterol, pH 7, or (iv) cholesterol, pH 5.7 media, before RNA was extracted for RNAseq analysis.Log 2 -fold change compares gene expression in each indicated condition to the 7H9, pH 7 condition.Genes marked in red had a log 2 -fold change � 0.6 between the two conditions compared (p<0.05,FDR<0.01 in both sets, with log 2 -fold change �1 in the single condition set).(B and C) Effect of acidic pH on Mtb response to cholesterol is concentration dependent.Log-phase Mtb was exposed to the indicated conditions, along with the control 7H9, pH 7 condition, for 4 hours, before RNA was extracted for qRT-PCR analysis.(E and F) Effect of cholesterol on Mtb response to acidic pH is concentration dependent.For all qRT-PCR data, fold change is as compared to the 7H9, pH 7 condition, and data are shown as means ± SEM from 3 experiments.The numerical data underlying the graphs shown in this figure are provided in S1 Data.https://doi.org/10.1371/journal.pgen.1011143.g004 Fig).Notably however, the simultaneous presence of cholesterol still resulted in an increase in the induction level of several tested genes, compared to the single acidic pH condition, in ΔphoPR Mtb (S2A Fig, compare open red bars to open black bars).The same phenomenon of an overall decrease in gene expression induction with the ΔphoPR Mtb mutant in both standard 7H9 or cholesterol media as a base was also observed with Cl -as the inducing signal instead of acidic pH (S2B Fig).Similarly, the continued increase in gene expression induction for a few tested genes in the simultaneous presence of cholesterol was also observed in the dual cholesterol/high [Cl -] condition compared to the single high [Cl -] condition (S2B Fig, compare open blue bars to open black bars).Complementation of the ΔphoPR Mtb mutant restored gene expression profiles to WT levels in all cases (S2 Fig).These findings suggest the involvement of additional regulators that contribute to the coordination of the acidic pH/Cl -response with Mtb response to cholesterol.
7 condition were very similar to those observed in the single cholesterol (pH 7) condition (compare open bars in Fig 5E to open bars in Fig 5D).Of note, augmentation in expression of acidic pH regulon genes by cholesterol was however still observed in the Δmce3R mutant as compared to WT Mtb (Fig 5F).Tests with the cell permeable pH-responsive dye 5-chloromethylfluorescein diacetate showed that maintenance of intrabacterial pH by Mtb in the presence of an acidic environment, in the absence or presence of cholesterol, was unaffected by mce3R deletion (S3 Fig).

Fig 5 .
Fig 5. Mce3R regulates Mtb response to cholesterol only in the context of acidic pH.Log-phase WT, Δmce3R and mce3R* (complemented strain) Mtb were exposed for 4 hours to (i) 7H9, pH 7, (ii) 7H9, pH 5.7, (iii) cholesterol, pH 7, or (iv) cholesterol, pH 5.7 media, before RNA was extracted for RNAseq (A-C) or qRT-PCR (D-F).For RNAseq data, log 2 -fold change compares gene expression in each indicated condition to the 7H9, pH 7 condition.Genes marked in red had a log 2 -fold change � 0.6 between Δmce3R and WT strains in the indicated condition (p<0.05,FDR<0.01 in both sets, with log 2 -fold change �1 in WT).For qRT-PCR data, fold change is as compared to the 7H9, pH 7 condition.Data are shown as means ± SEM from 3 experiments.p-values were obtained with an unpaired t-test with Welch's correction and Holm-Sidak multiple comparisons.N.S. not significant, ** p<0.01, *** p<0.001, **** p<0.0001.The numerical data underlying the graphs shown in this figure are provided in S1 Data.https://doi.org/10.1371/journal.pgen.1011143.g005

Fig 6 .
Fig 6.Mce3R deletion dampens Mtb response to K + in the presence of cholesterol.Log-phase WT, Δmce3R, and mce3R* (complemented strain) Mtb were exposed for 4 hours to (A) K + -free 7H9 or cholesterol media at pH 7, or (B) cholesterol or K + -free cholesterol media at pH 7, along with 7H9, pH 7 as the control condition.([K + ] = 7.35 mM in cholesterol, pH 7 medium.)Fold change is as compared to the 7H9, pH 7 condition in all cases.sigA was used as the control gene, and data are shown as means ± SEM from 3 experiments.For clarity of comparison, the cholesterol medium, pH 7 data in Fig 5D are shown in Fig 6B again.p-values were obtained with an unpaired t-test with Welch's correction and Holm-Sidak multiple comparisons.N.S. not significant, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.The numerical data underlying the graphs shown in this figure are provided in S1 Data.https://doi.org/10.1371/journal.pgen.1011143.g006 Changes in pH and [K + ], and differing presence of cholesterol, are all factors expected to occur during Mtb host colonization.While no differences in growth were observed between WT and Δmce3R Mtb in the various acidic pH, low [K + ], and cholesterol conditions used in the transcriptional analyses above (S4 Fig), the complexity of intact host cell or whole animal infection models can often reveal phenotypes not seen in the context of rich broth.To assess the importance of Mce3R during infection, we thus first infected bone marrow-derived macrophages (BMDMs) with WT, Δmce3R, or mce3R* Mtb.Given our findings of the importance of Mce3R in coordination of the cholesterol response of Mtb, these infections were also performed in BMDMs pre-treated with oleate to induce the formation of foamy macrophages [37,38], to determine whether infection in lipid-rich macrophages would differ from untreated BMDMs.As shown in Fig 7A, the Δmce3R mutant was attenuated for growth in BMDMs compared to WT and mce3R* Mtb, with a more pronounced effect in the context of foamy macrophage

Fig 7 .A
Fig 7. A Δmce3R Mtb mutant is attenuated for host colonization in the presence of lipids.(A) Δmce3R Mtb is attenuated for macrophage colonization.Murine bone marrow-derived macrophages untreated or pre-treated with oleate for 24 hours to induce foamy macrophages were infected with WT, Δmce3R, or mce3R* (complemented mutant) Mtb, and colony forming units (CFUs) tracked over time.Data are shown as means ± SD from 3 wells, representative of 3 independent experiments.p-values were obtained with an unpaired t-test with Welch's correction, comparing Δmce3R to WT Mtb in untreated macrophages (*) or foamy macrophages (#).*/# p<0.05, ** p<0.01.(B) Mtb is present in foamy macrophages 6 weeks post-infection in C3HeB/FeJ mice.C3HeB/FeJ mice were infected with Mtb constitutively expressing mCherry, and animals sacrificed at 2 or 6 weeks post-infection.Lungs were fixed and processed for confocal microscopy imaging.Images shown are z-slices from reconstructed 3D images.Nuclei are shown in grayscale (DAPI), all bacteria are marked in red (mCherry), f-actin is shown in blue (phalloidin), and lipid droplets are shown in green (Bodipy 493/503).Scale bar 10 μm.(C) Δmce3R Mtb is attenuated for colonization in a murine infection model.C3HeB/FeJ mice were infected with WT, Δmce3R, or mce3R* Mtb, and lung homogenates plated for CFUs 2 or 6 weeks post-infection.p-values were obtained with a Mann-Whitney statistical test.N.S. not significant, **p<0.01.The numerical data underlying the graphs shown in this figure are provided in S1 Data.https://doi.org/10.1371/journal.pgen.1011143.g007 ), consistent with this time point being prior to the formation of foamy macrophages (Fig 7B).In contrast, at the 6-week time point, the Δmce3R mutant exhibited a significantly reduced bacterial load compared to the WT and mce3R* Mtb strains (Fig 7C).Together, these results demonstrate that Mce3R plays an important role in Mtb growth in macrophages, with the absence of Mce3R resulting in a significantly reduced ability of Mtb to maintain persistent colonization of a whole animal host.
Fig 8B).It will be intriguing in future studies to explore the underlying mechanism enabling the triggering of Mce3R function only in the context of the concurrent presence of two signals versus one.Mce3R is a cytoplasmic protein and would not directly sense changes in external signals, although an increase in cholesterol uptake would result in increased levels of cholesterol within the bacterium.Of note, the affinity of Mce2R and Mce1R for binding to their target DNA have been reported (experimentally or computationally respectively) to change in the presence of long-chain fatty acids

Fig 8 .
Fig 8. Summary of the relationships between Mtb response to acidic pH, cholesterol, and low [K + ], and the role of Mce3R.A summary model and table of the interplay between acidic pH, cholesterol, and low [K + ] is shown in (A), with the effect of mce3R deletion on the various relationships shown in (B).Shaded boxes in (A) are the single conditions.As there was no effect on the response of Mtb to low [K + ] in the presence of acidic pH, the effect of mce3R deletion on this relationship was not tested here (shaded box in B). https://doi.org/10.1371/journal.pgen.1011143.g008