SummaryCyclic 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 CRPMt 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.

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.Tuberculosis (TB) is a leading cause of human death by an infectious agent, killing approximately 2 million people each year. Nearly one-third of the world's population is latently infected with the Mycobacterium tuberculosis bacillus, and 7 to 8 million new TB cases occur annually (49). A deadly synergy with the human immunodeficiency virus epidemic and an increasing proportion of drug-resistant cases contribute to TB's recent resurgence and complicate its treatment (7, 9). A better understanding of M. tuberculosis biology is needed to devise more effective treatments against TB. Gene regulation is critical for the interaction of the tubercle bacilli with their

Recurrent Clostridium difficile infection (CDI) is a consequence of intestinal dysbiosis and is particularly common following hematopoietic stem cell transplantation (HSCT). Fecal microbiota transplantation (FMT) is an effective method of treating CDI by correcting intestinal dysbiosis by passive transfer of healthy donor microflora. FMT has not been widely used in immunocompromised patients, including HSCT recipients, owing to concern for donor-derived infection. Here, we describe initial results of an FMT program for CDI at a US HSCT center. Seven HSCT recipients underwent FMT between February 2015 and February 2016. Mean time post HSCT was 635 days (25-75 interquartile range [IQR] 38-791). Five of the patients (71.4%) were on immunosuppressive therapy at FMT; 4 had required long-term suppressive oral vancomycin therapy because of immediate recurrence after antibiotic cessation. Stool donors underwent comprehensive health and behavioral screening and laboratory testing of serum and stool for 32 potential pathogens. FMT was administered via the naso-jejunal route in 6 of the 7 patients. Mean follow-up was 265 days (IQR 51-288). Minor post-FMT adverse effects included self-limited bloating and urgency. One patient was suspected of having post-FMT small intestinal bacterial overgrowth. No serious adverse events were noted and all-cause mortality was 0%. Six of 7 (85.7%) patients had no recurrence; 1 patient recurred at day 156 post FMT after taking an oral antibiotic and required repeat FMT, after which no recurrence has occurred. Diarrhea was improved in all patients and 1 patient with gastrointestinal graft-versus-host disease was able to taper off systemic immunosuppression after FMT. With careful donor selection and laboratory screening, FMT appears to be a safe and effective therapy for CDI in HSCT patients and may confer additional benefits. Larger studies are necessary to confirm safety and efficacy and explore other possible effects.

Mycobacterium tuberculosisTuberculosis (TB) remains a global epidemic, with one-third of the world's population infected, 9 million active cases, and over 2 million deaths annually (3). Increased drug resistance and a lethal synergism with human immunodeficiency virus exacerbate the morbidity and mortality already associated with Mycobacterium tuberculosis infection (7, 9). The molecular pathogenesis of M. tuberculosis is poorly understood, and better characterization of the mechanisms by which the tubercle bacillus responds to its host environment is needed if we are to control this deadly infection.Cyclic AMP (cAMP), generated by adenylate cyclase, is an important signaling molecule in many bacterial species and eukaryotic cells. cAMP-mediated gene regulation in Escherichia coli, a process which requires interaction with the cAMP receptor protein (CRP), has been well defined (16). Additional roles for cAMP in metabolism have been reported for many pathogens (2,6,8,13,18,19,23,30,36), and recent studies increasingly point to an important role for cAMP signaling in microbial pathogenesis. Vfr, a CRP homolog in Pseudomonas aeruginosa, coordinates the expression of a variety of virulenceassociated factors including type IV pili and the type III secretion system (35). Mutation of a cyclase-encoding gene (cya) in Vibrio vulnificus resulted in attenuation of the bacterium, with a 100-fold increase in the 50% lethal dose for mice (15). Pathogenic fungi also use the highly conserved cAMP signal transduction pathway to regulate cellular differentiation and pathogenesis (20,21,31).The potential significance of cAMP signaling in M. tuberculosis pathogenesis was first explored in an early study that reported elevated levels of cAMP in the cytoplasms of macrophages infected with M. microti (24). Those authors correlated such increased cAMP levels with impaired phagosome-lysosome fusion. However, little more work was done on this topic until an in silico study identified genes for 15 adenylate cyclases and 10 cNMP-binding proteins in the M. tuberculosis genome (27). At least 10 of these adenylate cyclases are functional, as demonstrated by in vitro biochemical assays (33). It was recently shown that cAMP signaling is likely to be an important global regulator of gene regulation in M. tuberculosis and also that Rv3676 (CRP Mt ) is a CRP-like cAMP-responsive transcription factor with a putative regulon of more than 100 genes (4,32,34). Deletion of Rv3676 caused impaired growth in laboratory medium, in bone marrow-derived macrophages, and in a mouse model of TB (32).CRP Mt and its ortholog in M. bovis are identical. However, the Mycobacterium bovis BCG Pasteur ortholog, which we refer to as CRP BCG , contains two amino acid differences, L47P and E178K, relative to the CRP Mt sequence. It was recently reported that the L47P change resulted in loss of cAMP binding and that the E178K substitution caused a DNA binding defect in an E. coli model system (34). The authors proposed a role for CRP BCG in M. bovis BCG's attenuation based...

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.