Mycobacterial phosphodiesterase Rv0805 is a virulence determinant and its cyclic nucleotide hydrolytic activity is required for propionate detoxification.

Cyclic AMP (cAMP) signaling is essential to Mycobacterium tuberculosis (Mtb) pathogenesis. However, the roles of phosphodiesterases (PDEs) Rv0805, and the recently identified Rv1339, in cAMP homeostasis and Mtb biology are unclear. We found that Rv0805 modulates Mtb growth within mice, macrophages and on host-associated carbon sources. Mycobacterium bovis BCG grown on a combination of propionate and glycerol as carbon sources showed high levels of cAMP and had a strict requirement for Rv0805 cNMP hydrolytic activity. Supplementation with vitamin B12 or spontaneous genetic mutations in the pta-ackA operon restored the growth of BCGΔRv0805 and eliminated propionate-associated cAMP increases. Surprisingly, reduction of total cAMP levels by ectopic expression of Rv1339 restored only 20% of growth, while Rv0805 complementation fully restored growth despite a smaller effect on total cAMP levels. Deletion of an Rv0805 localization domain also reduced BCG growth in the presence of propionate and glycerol. We propose that localized Rv0805 cAMP hydrolysis modulates activity of a specialized pathway associated with propionate metabolism, while Rv1339 has a broader role in cAMP homeostasis. Future studies will address the biological roles of Rv0805 and Rv1339, including their impacts on metabolism, cAMP signaling and Mtb pathogenesis.

A simple protein-based surrogate neutralization assay for SARS-CoV-2.

Most of the patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mount a humoral immune response to the virus within a few weeks of infection, but the duration of this response and how it correlates with clinical outcomes has not been completely characterized. Of particular importance is the identification of immune correlates of infection that would support public health decision-making on treatment approaches, vaccination strategies, and convalescent plasma therapy. While ELISA-based assays to detect and quantitate antibodies to SARS-CoV-2 in patient samples have been developed, the detection of neutralizing antibodies typically requires more demanding cell-based viral assays. Here, we present a safe and efficient protein-based assay for the detection of serum and plasma antibodies that block the interaction of the SARS-CoV-2 spike protein receptor binding domain (RBD) with its receptor, angiotensin-converting enzyme 2 (ACE2). The assay serves as a surrogate neutralization assay and is performed on the same platform and in parallel with an ELISA for the detection of antibodies against the RBD, enabling a direct comparison. The results obtained with our assay correlate with those of 2 viral-based assays, a plaque reduction neutralization test (PRNT) that uses live SARS-CoV-2 virus and a spike pseudotyped viral vector-based assay.

Chemical activation of adenylyl cyclase Rv1625c inhibits growth of Mycobacterium tuberculosis on cholesterol and modulates intramacrophage signaling.

Mycobacterium tuberculosis (Mtb) uses a complex 3', 5'-cyclic AMP (cAMP) signaling network to sense and respond to changing environments encountered during infection, so perturbation of cAMP signaling might be leveraged to disrupt Mtb pathogenesis. However, understanding of cAMP signaling pathways is hindered by the presence of at least 15 distinct adenylyl cyclases (ACs). Recently, the small molecule V-58 was shown to inhibit Mtb replication within macrophages and stimulate cAMP production in Mtb. Here we determined that V-58 rapidly and directly activates Mtb AC Rv1625c to produce high levels of cAMP regardless of the bacterial environment or growth medium. Metabolic inhibition by V-58 was carbon source dependent in Mtb and did not occur in Mycobacterium smegmatis, suggesting that V-58-mediated growth inhibition is due to interference with specific Mtb metabolic pathways rather than a generalized cAMP toxicity. Chemical stimulation of cAMP production by Mtb within macrophages also caused down regulation of TNF-α production by the macrophages, indicating a complex role for cAMP in Mtb pathogenesis. Together these studies describe a novel approach for targeted stimulation of cAMP production in Mtb, and provide new insights into the myriad roles of cAMP signaling in Mtb, particularly during Mtb's interactions with macrophages.

Characterization of a cAMP responsive transcription factor, Cmr (Rv1675c), in TB complex mycobacteria reveals overlap with the DosR (DevR) dormancy regulon.

Mycobacterium tuberculosis (Mtb) Cmr (Rv1675c) is a CRP/FNR family transcription factor known to be responsive to cAMP levels and during macrophage infections. However, Cmr's DNA binding properties, cellular targets and overall role in tuberculosis (TB) complex bacteria have not been characterized. In this study, we used experimental and computational approaches to characterize Cmr's DNA binding properties and identify a putative regulon. Cmr binds a 16-bp palindromic site that includes four highly conserved nucleotides that are required for DNA binding. A total of 368 binding sites, distributed in clusters among ~200 binding regions throughout the Mycobacterium bovis BCG genome, were identified using ChIP-seq. One of the most enriched Cmr binding sites was located upstream of the cmr promoter, and we demonstrated that expression of cmr is autoregulated. cAMP affected Cmr binding at a subset of DNA loci in vivo and in vitro, including multiple sites adjacent to members of the DosR (DevR) dormancy regulon. Our findings of cooperative binding of Cmr to these DNA regions and the regulation by Cmr of the DosR-regulated virulence gene Rv2623 demonstrate the complexity of Cmr-mediated gene regulation and suggest a role for Cmr in the biology of persistent TB infection.

Role of intragenic binding of cAMP responsive protein (CRP) in regulation of the succinate dehydrogenase genes Rv0249c-Rv0247c in TB complex mycobacteria.

Bacterial pathogens adapt to changing environments within their hosts, and the signaling molecule adenosine 3', 5'-cyclic monophosphate (cAMP) facilitates this process. In this study, we characterized in vivo DNA binding and gene regulation by the cAMP-responsive protein CRP in M. bovis BCG as a model for tuberculosis (TB)-complex bacteria. Chromatin immunoprecipitation followed by deep-sequencing (ChIP-seq) showed that CRP associates with ∼900 DNA binding regions, most of which occur within genes. The most highly enriched binding region was upstream of a putative copper transporter gene (ctpB), and crp-deleted bacteria showed increased sensitivity to copper toxicity. Detailed mutational analysis of four CRP binding sites upstream of the virulence-associated Rv0249c-Rv0247c succinate dehydrogenase genes demonstrated that CRP directly regulates Rv0249c-Rv0247c expression from two promoters, one of which requires sequences intragenic to Rv0250c for maximum expression. The high percentage of intragenic CRP binding sites and our demonstration that these intragenic DNA sequences significantly contribute to biologically relevant gene expression greatly expand the genome space that must be considered for gene regulatory analyses in mycobacteria. These findings also have practical implications for an important bacterial pathogen in which identification of mutations that affect expression of drug target-related genes is widely used for rapid drug resistance screening.

Cyclic AMP Signaling in Mycobacteria.

All cells must adapt to changing conditions, and many use cyclic AMP (cAMP) as a second messenger to sense and respond to fluctuations in their environment. cAMP is made by adenylyl cyclases (ACs), and mycobacteria have an unusually large number of biochemically distinct ACs. cAMP is important for gene regulation in mycobacteria, and the ability to secrete cAMP into host macrophages during infection contributes to Mycobacterium tuberculosis pathogenesis. This article discusses the many roles of cAMP in mycobacteria and reviews what is known about the factors that contribute to production, destruction, and utilization of this important signal molecule. Special emphasis is placed on cAMP signaling in M. tuberculosis complex bacteria and its importance to M. tuberculosis during host infection.

The myriad roles of cyclic AMP in microbial pathogens: from signal to sword.

All organisms must sense and respond to their external environments, and this signal transduction often involves second messengers such as cyclic nucleotides. One such nucleotide is cyclic AMP, a universal second messenger that is used by diverse forms of life, including mammals, fungi, protozoa and bacteria. In this review, we discuss the many roles of cAMP in bacterial, fungal and protozoan pathogens and its contributions to microbial pathogenesis. These roles include the coordination of intracellular processes, such as virulence gene expression, with extracellular signals from the environment, and the manipulation of host immunity by increasing cAMP levels in host cells during infection.

Dysregulation of serine biosynthesis contributes to the growth defect of a Mycobacterium tuberculosis crp mutant.

Mycobacterium tuberculosis CRP(Mt), encoded by Rv3676 (crp), is a CRP-like transcription factor that binds with the serC-Rv0885 intergenic region. In the present study, we evaluated CRP(Mt) 's regulation of serC and Rv0885 in M. tuberculosis and M. bovis BCG, using site-specific mutagenesis, promoter fusions and reverse-transcriptase PCR (RT-PCR). The CRP(Mt) binding site was required for full expression of serC and Rv0885, and expression of both genes was reduced in M. tuberculosis and M. bovis BCG crp mutants. These data show that CRP(Mt) binding directly activates both serC and Rv0885 expression. M. tuberculosis serC restored the ability of an Escherichia coli serC mutant to grow in serine-dropout medium, demonstrating that M. tuberculosis serC encodes a phosphoserine aminotransferase. Serine supplementation, or overexpression of serC, accelerated the growth of M. tuberculosis and M. bovis BCG crp mutants in mycomedium, but not within macrophages. These results establish a role for CRP(Mt) in the regulation of amino acid biosynthesis, and show that reduced serine production contributes to the slow-growth phenotype of M. tuberculosis and M. bovis BCG crp mutants in vitro. Restoration of serine biosynthesis by serC expression will facilitate identification of additional CRP(Mt)-regulated factors required by M. tuberculosis during macrophage and host infection.

Cyclic AMP signalling in mycobacteria: redirecting the conversation with a common currency.

cAMP is an ancient second messenger, and is used by many organisms to regulate a wide range of cellular functions. Mycobacterium tuberculosis complex bacteria are exceptional in that they have genes for at least 15 biochemically distinct adenylyl cyclases, the enzymes that generate cAMP. cAMP-associated gene regulation within tubercle bacilli is required for their virulence, and secretion of cAMP produced by M. tuberculosis bacteria into host macrophages disrupts the host's immune response to infection. In this review, we discuss recent advances in our understanding of the means by which cAMP levels are controlled within mycobacteria, the importance of cAMP to M. tuberculosis during host infection, and the role of cAMP in mycobacterial gene regulation. Understanding the myriad aspects of cAMP signalling in tubercle bacilli will establish new paradigms for cAMP signalling, and may contribute to new approaches for prevention and/or treatment of tuberculosis disease.

cAMP levels within Mycobacterium tuberculosis and Mycobacterium bovis BCG increase upon infection of macrophages.

Adenosine 3',5'-cyclic monophosphate (cAMP)-mediated signal transduction is common in both prokaryotes and eukaryotes, and several bacterial pathogens modulate cAMP signaling pathways of their mammalian hosts during infection. In this study, cAMP levels associated with Mycobacterium tuberculosis and Mycobacterium bovis BCG were measured during macrophage infection. cAMP levels within both bacteria increased c. 50-fold during infection of J774.16 macrophages, relative to the cAMP levels within bacteria incubated in tissue culture media alone. cAMP levels also increased within the macrophage cytoplasm upon uptake of live, but not dead, mycobacteria. The presence of albumin in the absence of oleic acid significantly decreased cAMP secretion and production by both M. tuberculosis and M. bovis BCG. These results suggest that cAMP signaling plays a role in the interaction of tuberculosis-complex mycobacteria with macrophages during infection, and that albumin may be a physiological indicator differentiating host environments during infection.

Rv1675c (cmr) regulates intramacrophage and cyclic AMP-induced gene expression in Mycobacterium tuberculosis-complex mycobacteria.

Cyclic AMP (cAMP) has recently been shown to be a global regulator of gene expression in Mycobacterium tuberculosis (Mtb). In this study we identified a new cAMP-associated regulon in Mtb and Mycobacterium bovis BCG, which is distinct from the previously described CRP(Mt) regulon. Proteomic comparison of wild-type M. bovis BCG with a Rv1675c (cmr) knockout strain showed dysregulated expression of four previously identified proteins encoded by the cAMP-induced genes (cAIGs) mdh, groEL2, Rv1265 and PE_PGRS6a. Regulated expression of these four cAIGs also occurred during macrophage infection, and this regulation required cmr in both Mtb and M. bovis BCG. Purified His-Cmr bound to the DNA sequences upstream of three cAIGs (mdh, groEL2, Rv1265) in electrophoretic mobility shift assays, suggesting direct regulation of these genes by Cmr. We also found that low pH stimulated cAMP production in both Mtb and M. bovis BCG, but broadly affected cAIG regulation only in M. bovis BCG. These studies identify Cmr as a transcription factor that regulates cAIGs within macrophages, and suggest that multiple factors affect cAMP-associated gene regulation in tuberculosis-complex mycobacteria. cAMP signalling and Cmr-mediated gene regulation during Mtb infection of macrophages may have implications for TB pathogenesis.

Characterization of Mycobacterium tuberculosis Rv3676 (CRPMt), a cyclic AMP receptor protein-like DNA binding protein.

Little is known about cyclic AMP (cAMP) function in Mycobacterium tuberculosis, despite its ability to encode 15 adenylate cyclases and 10 cNMP-binding proteins. M. tuberculosis Rv3676, which we have designated CRP(Mt), is predicted to be a cAMP-dependent transcription factor. In this study, we characterized CRP(Mt)'s interactions with DNA and cAMP, using experimental and computational approaches. We used Gibbs sampling to define a CRP(Mt) DNA motif that resembles the cAMP receptor protein (CRP) binding motif model for Escherichia coli. CRP(Mt) binding sites were identified in a total of 73 promoter regions regulating 114 genes in the M. tuberculosis genome, which are being explored as a regulon. Specific CRP(Mt) binding caused DNA bending, and the substitution of highly conserved nucleotides in the binding site resulted in a complete loss of binding to CRP(Mt). cAMP enhanced CRP(Mt)'s ability to bind DNA and caused allosteric alterations in CRP(Mt) conformation. These results provide the first direct evidence for cAMP binding to a transcription factor in M. tuberculosis, suggesting a role for cAMP signal transduction in M. tuberculosis and implicating CRP(Mt) as a cAMP-responsive global regulator.

Identification of cyclic AMP-regulated genes in Mycobacterium tuberculosis complex bacteria under low-oxygen conditions.

Mycobacterium tuberculosis is the etiological agent of tuberculosis (TB), which kills approximately 2 million people a year despite current treatment options. A greater understanding of the biology of this bacterium is needed to better combat TB disease. The M. tuberculosis genome encodes as many as 15 adenylate cyclases, suggesting that cyclic AMP (cAMP) has an important, yet overlooked, role in mycobacteria. This study examined the effect of exogenous cAMP on protein expression in Mycobacterium bovis BCG grown under hypoxic versus ambient conditions. Both shaking and shallow standing cultures were examined for each atmospheric condition. Different cAMP-dependent changes in protein expression were observed in each condition by two-dimensional gel electrophoresis. Shaking low-oxygen cultures produced the most changes (12), while standing ambient conditions showed the fewest (2). Five upregulated proteins, Rv1265, Rv2971, GroEL2, PE_PGRS6a, and malate dehydrogenase, were identified from BCG by mass spectrometry and were shown to also be regulated by cAMP at the mRNA level in both M. tuberculosis H37Rv and BCG. To our knowledge, these data provide the first direct evidence for cAMP-mediated gene regulation in TB complex mycobacteria.

Identification of a Mycobacterium tuberculosis putative classical nitroreductase gene whose expression is coregulated with that of the acr aene within macrophages, in standing versus shaking cultures, and under low oxygen conditions.

Tuberculosis remains a leading killer worldwide, and new approaches for its treatment and prevention are urgently needed. This effort will benefit greatly from a better understanding of gene regulation in Mycobacterium tuberculosis, particularly with respect to this pathogen's response to its host environment. We examined the behavior of two promoters from the divergently transcribed M. tuberculosis genes acr/hspX/Rv2031c (alpha-crystallin homolog) and Rv2032/acg (acr-coregulated gene) by using a promoter-GFP fusion assay in Mycobacterium bovis BCG. We found that Rv2032 is a novel macrophage-induced gene whose expression is coregulated with that of acr. Relative levels of intracellular induction for both promoters were significantly affected by shallow standing versus shaking bacterial culture conditions prior to macrophage infection, and both promoters were strongly induced under low oxygen conditions. Deletion analyses showed that DNA sequences within a 43-bp region were required for expression of these promoters under all conditions. Multiple sequence alignment and database searches performed with PROBE indicated that Rv2032 is one of eight M. tuberculosis genes of previously unknown function that belong to an unusual superfamily of classical nitroreductases, which may have a role for bacteria within the host environment. These findings show that mycobacterial culture conditions can greatly influence the results and interpretation of subsequent gene regulation experiments. We propose that these differences might be exploited for dissection of the regulatory factors that affect mycobacterial gene expression within the host.