Mycobacterium tuberculosis, the causative agent of pulmonary tuberculosis, encounters diverse environmental conditions during infection, persistence, and transmission of the disease (Ehrt et al., 2018; Ernst, 2012; Russell, 2011). However, the pathogen is exceptionally capable of adjusting and surviving within diverse host environments. Mycobacterial adaptive response to different stages of infection is achieved via fine-tuning of regulation of gene expression using an extensive repertoire of more than 100 transcriptional regulators, 11 two-component systems, 6-serine-threonine protein kinases, and 13 alternative sigma factors, suggesting that a very complex transcriptional program is important for M. tuberculosis pathogenesis. While the regulation of gene expression as a consequence of interaction of tubercle bacilli with its immediate environment remains critically important (Rohde et al., 2007), defining these regulatory pathways represents a major challenge in the field.

3′,5′-cyclic adenosine monophosphate (cAMP), one of the most widely used second messengers, impacts a wide range of cellular responses in microbial pathogens, including M. tuberculosis (McDonough and Rodriguez, 2011). The bacterial genome encodes at least 15 adenylate cyclases (ACs), including one of the ACs (Rv0386) that is required for virulence (Agarwal et al., 2009), and multiple cAMP-regulated effector proteins (Banerjee et al., 2015; Johnson and McDonough, 2018; McDonough and Rodriguez, 2011; Shenoy and Visweswariah, 2006). cAMP levels are elevated upon infection of macrophages by pathogenic mycobacterium (Bai et al., 2009), and addition of exogenous cAMP was shown to influence mycobacterial protein expression (Gazdik and McDonough, 2005). While intra-bacterial cAMP responds to macrophage environment and carries out specific functions like gene expression and protein function under host-related conditions (Bai et al., 2011; Bai et al., 2009; Johnson et al., 2017; Johnson and McDonough, 2018; Knapp and McDonough, 2014), secreted cAMP controls macrophage signaling (Agarwal et al., 2009; Agarwal et al., 2006; Gazdik et al., 2009; Johnson et al., 2017; Nambi et al., 2013; Ranganathan et al., 2016; Rittershaus et al., 2018). cAMP signaling remains essential to M. tuberculosis pathogenesis. Agarwal and colleagues had shown that a burst in synthesis of cAMP upon infection of macrophages improved bacterial survival by interfering with host signaling pathways (Agarwal et al., 2009). In keeping with this, anti-tubercular compounds that interfere with mycobacterial cAMP levels also impact intracellular growth of mycobacteria (Bai et al., 2011; Johnson et al., 2017; Knapp et al., 2015; Nambi et al., 2013; Rittershaus et al., 2018; VanderVen et al., 2015; Wilburn et al., 2022). Together, these results underscore the significance of cAMP signalling in mycobacteria.

cAMP signaling is controlled at the transcriptional level. Previous in silico studies had identified 2 out of 10 predicted nucleotide-binding proteins of M. tuberculosis as members of the CRP/FNR superfamily of transcriptional regulators (McCue et al., 2000). These are CRP (cyclic AMP receptor protein), encoded by rv3676 and CMR (cAMP macrophage regulator), encoded by rv1675c, respectively. Of these two, CRP becomes activated upon cAMP binding and functions as a global regulator of ~100 genes (Agarwal et al., 2006; Bai et al., 2005; Rickman et al., 2005). Consequently, deletion of the crp locus significantly impairs mycobacterial growth and attenuates virulence of the bacilli in a mouse model (Rickman et al., 2005). In contrast, CMR is necessary for regulated expression of genes involved in virulence and persistence, including members of the dormancy regulon (Gazdik et al., 2009; Ranganathan et al., 2016; Smith et al., 2017). These results strongly suggest that cAMP homeostasis, that is, a balance between cAMP synthesis by ACs and cAMP degradation by phosphodiesterases (PDEs), contributes to rapid adaptive response of mycobacteria in a hostile intracellular environment (Johnson and McDonough, 2018; McDonough and Rodriguez, 2011). However, very little is known about the underlying mechanisms of regulation of mycobacterial cAMP level.

M. tuberculosis encounters a hostile environment within the host. Among the hostile conditions are the acidic pH stress and exposure to host immune effectors such as NO (Nathan and Shiloh, 2000; Rustad et al., 2009; Wayne and Sohaskey, 2001). A growing body of evidence connects virulence-associated mycobacterial phoP locus with varying environmental conditions, including acid stress (Abramovitch et al., 2011; Bansal et al., 2017; Tan et al., 2013), heat-shock (Sevalkar et al., 2019; Singh et al., 2014), and integration of acid stress response to redox homeostasis (Baker et al., 2019; Baker et al., 2014; Goar et al., 2022). Disruption of phoP, the gene encoding the response regulator of the PhoPR two-component signal transduction system (Gupta et al., 2006), significantly reduces in vivo multiplication of the bacilli (Walters et al., 2006), suggesting that PhoPR remains essential for virulence (Pérez et al., 2001). Moreover, a mutant lacking this system shows a significantly lowered synthesis of cell wall components diacyltrehaloses, polyacyltrehaloses, and sulfolipids, specific to pathogenic mycobacterial species (Gonzalo Asensio et al., 2006; Goyal et al., 2011; Walters et al., 2006). In fact, the significant attenuation of phoPR deletion strain forms the basis of the mutant being considered in trials as a vaccine strain (Arbues et al., 2013). Recent transcriptomic analyses revealed that approximately 2% of the H37Rv genome is regulated by PhoP (Solans et al., 2014; Walters et al., 2006), and mycobacterial gene expression in response to acidic pH significantly overlaps with the PhoP regulon (Rohde et al., 2007). Consistent with this, a large subset of PhoPR-regulated low-pH-inducible genes are induced immediately following M. tuberculosis phagocytosis and remain induced during macrophage infection (Gonzalo Asensio et al., 2006; Martin et al., 2006; Pérez et al., 2001; Walters et al., 2006). Additional evidence, coupled with more recent results, suggests that during the onset of macrophage infection PhoPR activation is linked to acidic pH and the available carbon source, suggesting a physiological link between pH, carbon source, and macrophage pathogenesis (Baker et al., 2019).

In this study, we hypothesized that intra-mycobacterial cAMP level could be determined by PhoP since the major regulator has been implicated in the regulation of bacterial responses against numerous stress conditions, many of which function as signals to activate cAMP synthesizing diverse ACs (Knapp and McDonough, 2014). Our results connect virulence regulator PhoP with intra-mycobacterial cAMP level. We discovered that PhoP regulates the expression of cAMP-specific phosphodiesterase rv0805, which hydrolyzes cAMP. To further probe the regulation, we demonstrate that under the condition that activates PhoP-PhoR system, PhoP in a PhoR-dependent manner represses the transcription of rv0805 through direct DNA binding at the upstream regulatory region. These observations account for a consistently lower level of cAMP in a PhoPR-deleted M. tuberculosis H37Rv (phoPR-KO) relative to the WT bacilli and establishes the molecular mechanism of regulation of cAMP level, absence of which strikingly impacts phagosome maturation, and reduces mycobacterial survival within macrophages and mice. Together, the newly identified mechanism of regulation of cAMP level allows intra-phagosomal survival and growth program of mycobacteria.

A number of studies suggest that conditions associated with host environment like low pH and macrophage interactions often influence mycobacterial cAMP levels (Bai et al., 2009; Gazdik and McDonough, 2005). Although many bacterial pathogens modulate host cell cAMP levels as a common strategy, the mechanism of regulation of mycobacterial cAMP-level remains unknown. In this study, we sought to investigate whether PhoP, a master regulator implicated in controlling diverse mycobacterial stress response, regulates mycobacterial cAMP level. We find that under normal conditions as well as under carefully controlled single stress conditions, phoPR-KO shows a significantly lower level of cAMP relative to the WT bacilli (Figure 1A), and complementation of the mutant restored cAMP level. To investigate the mechanism, we next probed regulation of ACs and PDEs (Figure 2) and demonstrated that PhoP functions as a major repressor of rv0805, encoding cAMP-specific PDE. Indeed, this newly identified rv0805 regulation, coupled with a recent discovery that phosphodiesterase activity of Rv0805 controls propionate detoxification (McDowell et al., 2023), fits well with and explains the previously puzzling in vivo observation by Abramovitch et al., 2011 that PhoP-controlled aprABC locus is associated with the regulation of genes of carbon and propionate metabolism.

Although a large number of ACs are present in M. tuberculosis genome, a class III metallo-phosphoesterase Rv0805 was earlier considered the only PDE, specific for mycobacterial cAMP. However, a recent study has identified an atypical class II PDE Rv1339, which upon overexpression reduces cAMP level and contributes to antibiotic sensitivity (Thomson et al., 2022). While the functional role of Rv1339 in M. tuberculosis is yet to be understood, crystal structure and biochemical evidence suggest that dimeric Rv0805 is stabilized by the presence of a divalent cation and remains catalytically active on a broad range of linear and cyclic PDE substrates in vitro (Keppetipola and Shuman, 2008; Shenoy et al., 2007; Shenoy et al., 2005). More recently, cyclic nucleotide hydrolytic activity of mycobacterial Rv0805 has been implicated in propionate detoxification (McDowell et al., 2023). However, the mechanism of regulation of Rv0805 and its effect on mycobacterial cAMP level remained unknown before the present study.

To examine biological significance of PhoP-dependent Rv0805 expression, we studied rv0805 expression under acidic conditions of growth as phoPR system is induced under acidic pH both in vitro and in macrophages (Abramovitch et al., 2011; Bansal et al., 2017). A significantly higher repression of rv0805 expression under acidic pH relative to normal conditions is consistent with the activation of PhoP and subsequent repression of rv0805 (Figure 3). These results further suggest that effective mitigation of stress by mycobacteria possibly requires a higher cAMP level for survival under intra-phagosomal environment. In keeping with these results, we find that (a) P~PhoP binds to rv0805 regulatory region (Figure 2—figure supplement 1) and (b) PhoP-dependent rv0805 expression requires PhoR (Figure 3A and B), the cognate kinase which activates PhoP in a signal-dependent manner (Gupta et al., 2006; Singh et al., 2023). These results account for a consistently lower level of cAMP in phoPR-KO relative to the WT bacilli. Notably, except recently reported PDE Rv1339, Rv0805 has been known as the only cAMP-specific PDE present in the slow-growing pathogenic mycobacteria and its closely related species (Matange, 2015; Shenoy et al., 2007; Shenoy et al., 2005), and Rv1339 expression does not appear to be regulated by the phoP locus (Figure 2). Thus, the above results showing PhoP-dependent repression of rv0805 activity likely represent the most critical step of regulation of mycobacterial cAMP level under stress. In this connection, our recent results that PhoP interacts with cAMP receptor protein, CRP, and a complex of two interacting regulators control expression of virulence determinants (Khan et al., 2022) invite speculation of a complex regulatory control of cAMP-responsive mycobacterial physiology.

As one might argue that PhoP deletion and rv0805 overexpression could be unrelated and independent events, we constructed phoP and rv0805 knockdown mutants to further investigate the PhoP-Rv0805-cAMP pathway. Our objective was to probe regulation of expression (Figure 3D) and examine the impact on mycobacterial cAMP (Figure 3E). phoP-KD significantly elevated rv0805 expression; however, phoP expression remains unaffected in rv0805-KD (Figure 3D). While elevated rv0805 expression in phoP-KD reduces cAMP level, understandably cAMP level is elevated in rv0805-KD mutant (Figure 3E). These results integrate PhoP-dependent rv0805 repression with mycobacterial cAMP level, suggesting how phoP-deletion or knockdown, at least in part, mimics Rv0805 overexpression. These considerations take on more significance given the fact that these two events have similar consequences on relevant strains with respect to stress tolerance and survival in cellular and animal models. Thus, our results suggest that ectopic expression of rv0805 is functionally equivalent to deletion of the phoP locus. This observation is in apparent conflict with a previous work by Matange et al., 2013, suggesting a cAMP-independent transcriptional response in rv0805 overexpressing M. tuberculosis H37Rv. Although both studies were performed with rv0805 overexpressing bacilli, the fact that important differences in the expression of PDEs in this study (Matange et al., 2013) and in our assays – yielding significantly different levels of rv0805 expression – most likely account for this discrepancy. While we cannot completely rule out the possibility of cleavage of other cyclic nucleotide(s) by Rv0805 (Keppetipola and Shuman, 2008; Shenoy et al., 2007; Shenoy et al., 2005) impacting our results, consistent with a previous study, our results correlate rv0805 expression with intra-mycobacterial cAMP level (Agarwal et al., 2009). Further, our data on the effect of expression of cyclic nucleotide-specific PDE Rv0805 or its inactive mutant (Rv0805M) correlate well with enzyme activities of the corresponding PDEs on mycobacterial cAMP levels (Figure 3C). Thus, we infer that PhoP-dependent regulation of rv0805 is a critical regulator of intra-mycobacterial cAMP level.

Our experiments to understand the physiological significance of PhoP-dependent repression of rv0805 expression uncover a comparable stress tolerance of WT-Rv0805 and phoPR-KO (significantly reduced relative to WT) (Figure 4). These results are consistent with the notion that cAMP level, at least in part, accounts for mycobacterial stress response. Along the line, WT-Rv0805 displayed a reduced ability to inhibit phagosome-lysosome fusion like phoPR-KO (Figure 4). Further, we show that WT-Rv0805, unlike the WT bacilli or WT-Rv0805M, shows a significantly reduced intracellular growth in mice as that of phoPR-KO (Figure 5). Thus, these results are of fundamental significance to establish that PhoP contributes to the maintenance of cAMP level and integrates it to the mechanisms of mycobacterial stress tolerance and intracellular survival. Together, we identify a novel mycobacterial pathway as a therapeutic target and provide yet another example of an intimate link between bacterial physiology and intracellular survival of the tubercle bacilli.

The results reported here are presented schematically in Figure 6. In summary, upon sensing low acidic pH as a signal PhoR activates PhoP, P~PhoP binds to rv0805 upstream regulatory region and functions as a specific repressor of Rv0805. Therefore, we observed (a) a reproducibly lower level of cAMP in phoPR-KO relative to WT-H37Rv, (b) a significantly reduced expression of rv0805 in WT-H37Rv, grown under acidic pH relative to normal conditions, and (c) comparable cAMP levels in phoPR-KO and WT-Rv0805. This is why the two strains remain ineffective to mount an appropriate stress response, most likely due to their inability to coordinate regulation of gene expression because of dysregulation of intra-mycobacterial cAMP level. However, without uncoupling regulatory control of PhoPR and rv0805 expression, we cannot confirm that dysregulation of cAMP level accounts for virulence attenuation of phoPR-KO. Given the fact that rv0805-depleted M. tuberculosis is growth attenuated in vivo (McDowell et al., 2023), paradoxically ectopic expression of rv0805 leads to dysregulated metabolic adaptation, thereby resulting in reduced stress tolerance and intracellular survival.

Figure 6

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All data generated or analysed during this study are included in the manuscript and supporting files; source data files have been provided for the figures.

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Author details

1. Hina Khan

   CSIR, Institute of Microbial Technology, Chandigarh, India

   Present address

   Department of Physiology, Michigan State University, Interdisciplinary Science and Technology Building, Lansing, United States

   Contribution

   Conceptualization, Data curation, Formal analysis

   Competing interests

   No competing interests declared

2. Partha Paul

   CSIR, Institute of Microbial Technology, Chandigarh, India

   Contribution

   Conceptualization, Data curation, Formal analysis

   Contributed equally with

   Harsh Goar and Bhanwar Bamniya

   Competing interests

   No competing interests declared

3. Harsh Goar

   CSIR, Institute of Microbial Technology, Chandigarh, India

   Present address

   Department of Medicine, Division of Hematology-Oncology UT Southwestern Medical Center, Dallas, United States

   Contribution

   Conceptualization, Data curation, Formal analysis

   Contributed equally with

   Partha Paul and Bhanwar Bamniya

   Competing interests

   No competing interests declared

4. Bhanwar Bamniya

   1. CSIR, Institute of Microbial Technology, Chandigarh, India
   2. Academy of Scientific and Innovative Research, Ghaziabad, India

   Contribution

   Data curation, Formal analysis

   Contributed equally with

   Partha Paul and Harsh Goar

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0009-0003-6467-678X

5. Navin Baid

   CSIR, Institute of Microbial Technology, Chandigarh, India

   Present address

   Department of Biosciences and Bioengineering Indian Institute of Technology Bombay, Mumbai, India

   Contribution

   Data curation, Formal analysis

   Competing interests

   No competing interests declared

6. Dibyendu Sarkar

   1. CSIR, Institute of Microbial Technology, Chandigarh, India
   2. Academy of Scientific and Innovative Research, Ghaziabad, India

   Contribution

   Conceptualization, Resources, Formal analysis, Supervision, Investigation, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   dibyendu@imtech.res.in

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6499-177X

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