Detection of cyclic-di-adenosine monophosphate (c-di-AMP), a bacterial second messenger, by the host cytoplasmic surveillance pathway (CSP) is known to elicit Type I interferon responses critical for antimicrobial defense1–3. However, the mechanisms and role of c-di-AMP signaling in Mycobacterium tuberculosis virulence remain unclear. Here we show that resistance to tuberculosis (TB) requires CSP-mediated detection of c-di-AMP produced by M. tuberculosis and that levels of c-di-AMP modulate the fate of infection. We found that a di-adenylate cyclase (disA or dacA)4 over-expressing M. tuberculosis strain that secretes excess c-di-AMP activates the interferon regulatory factor (IRF) pathway with enhanced levels of IFN-β, elicits increased macrophage autophagy, and exhibits significant attenuation in mice. We show that c-di-AMP-mediated IFN-β induction during M. tuberculosis infection requires stimulator of interferon genes (STING)5-signaling. We observed that c-di-AMP induction of IFN-β is independent of the cytosolic nucleic acid receptor cyclic-GMP-AMP (cGAMP) synthase (cGAS)6–7, but cGAS nevertheless contributes substantially to the overall IFN-β response to M. tuberculosis infection. In sum, our results reveal c-di-AMP to be a key mycobacterial pathogen associated molecular pattern (PAMP) driving host Type I IFN responses and autophagy. These findings suggest that modulating the levels of this small molecule may lead to novel immunotherapeutic strategies against TB.

Mycobacterium tuberculosis infection leads to cytosolic release of the bacterial cyclic dinucleotide (CDN) c-di-AMP and a host-generated CDN, cGAMP, both of which trigger type I interferon (IFN) expression in a STING-dependent manner. Here we report that M. tuberculosis has developed a mechanism to inhibit STING activation and the type I IFN response via the bacterial phosphodiesterase (PDE) CdnP, which mediates hydrolysis of both bacterial-derived c-di-AMP and host-derived cGAMP. Mutation of cdnP attenuates M. tuberculosis virulence, as does loss of a host CDN PDE known as ENPP1. CdnP is inhibited by both US Food and Drug Administration (FDA)-approved PDE inhibitors and nonhydrolyzable dinucleotide mimetics specifically designed to target the enzyme. These findings reveal a crucial role of CDN homeostasis in governing the outcome of M. tuberculosis infection as well as a unique mechanism of subversion of the host's cytosolic surveillance pathway (CSP) by a bacterial PDE that may serve as an attractive antimicrobial target.

The successful establishment and maintenance of a bacterial infection depends on the pathogen’s ability to subvert the host cell’s defense response and successfully survive, proliferate, or persist within the infected cell. To circumvent host defense systems, bacterial pathogens produce a variety of virulence factors that potentiate bacterial adherence and invasion and usurp host cell signaling cascades that regulate intracellular microbial survival and trafficking. Mycobacterium tuberculosis, probably one of the most successful pathogens on earth, has coexisted with humanity for centuries, and this intimate and persistent connection between these two organisms suggests that the pathogen has evolved extensive mechanisms to evade the human immune system at multiple levels. While some of these mechanisms are mediated by factors released by M. tuberculosis, others rely on host components that are hijacked to prevent the generation of an effective immune response thus benefiting the survival of M. tuberculosis within the host cell. Here, we describe several of these mechanisms, with an emphasis on the cyclic nucleotide signaling and subversion of host responses that occur at the intracellular level when tubercle bacilli encounter macrophages, a cell that becomes a safe-house for M. tuberculosis although it is specialized to kill most microbes.

BackgroundThe variable efficacy (0–80%) of Mycobacterium bovis Bacille Calmette Guréin (BCG) vaccine against adult tuberculosis (TB) necessitates development of alternative vaccine candidates. Development of recombinant BCG (rBCG) over-expressing promising immunodominant antigens of M. tuberculosis represents one of the potential approaches for the development of vaccines against TB.Methods/Principal FindingsA recombinant strain of BCG - rBCG85C, over expressing the antigen 85C, a secretory immuno-dominant protein of M. tuberculosis, was evaluated for its protective efficacy in guinea pigs against M. tuberculosis challenge by aerosol route. Immunization with rBCG85C resulted in a substantial reduction in the lung (1.87 log10, p<0.01) and spleen (2.36 log10, p<0.001) bacillary load with a commensurate reduction in pathological damage, when compared to the animals immunized with the parent BCG strain at 10 weeks post-infection. rBCG85C continued to provide superior protection over BCG even when post-challenge period was prolonged to 16 weeks. The cytokine profile of pulmonary granulomas revealed that the superior protection imparted by rBCG85C was associated with the reduced levels of pro-inflammatory cytokines - interleukin (IL)-12, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, moderate levels of anti-inflammatory cytokine - transforming growth factor (TGF)-β along with up-regulation of inducible nitric oxide synthase (iNOS). In addition, the rBCG85C vaccine induced modulation of the cytokine levels was found to be associated with reduced fibrosis and antigen load accompanied by the restoration of normal lung architecture.Conclusions/SignificanceThese results clearly indicate the superiority of rBCG85C over BCG as a promising prophylactic vaccine against TB. The enduring protection observed in this study gives enough reason to postulate that if an open-ended study is carried out with low dose of infection, rBCG85C vaccine in all likelihood would show enhanced survival of guinea pigs.

Pulmonary infections often cause spatially diffuse and multi-focal radiotracer uptake in positron emission tomography (PET) images, which makes accurate quantification of the disease extent challenging. Image segmentation plays a vital role in quantifying uptake due to the distributed nature of immuno-pathology and associated metabolic activities in pulmonary infection, specifically tuberculosis (TB). For this task, thresholding-based segmentation methods may be better suited over other methods; however, performance of the thresholding-based methods depend on the selection of thresholding parameters, which are often suboptimal. Several optimal thresholding techniques have been proposed in the literature, but there is currently no consensus on how to determine the optimal threshold for precise identification of spatially diffuse and multi-focal radiotracer uptake. In this study, we propose a method to select optimal thresholding levels by utilizing a novel intensity affinity metric within the affinity propagation clustering framework. We tested the proposed method against 70 longitudinal PET images of rabbits infected with TB. The overall dice similarity coefficient between the segmentation from the proposed method and two expert segmentations was found to be 91.25 ± 8.01% with a sensitivity of 88.80 ± 12.59% and a specificity of 96.01 ± 9.20%. High accuracy and heightened efficiency of our proposed method, as compared to other PET image segmentation methods, were reported with various quantification metrics.

Background Stimulator of interferon genes (STING) is a key cytosolic receptor for small nucleotides and plays a key role in anticancer and antiviral immunity. Cyclic dinucleotide STING agonists may comprise a novel class of vaccine adjuvants capable of inducing cellular immune responses and protective efficacy against intracellular pathogens. Methods We generated a recombinant Bacillus Calmette-Guérin ([BCG] BCG-disA-OE) that overexpresses the endogenous mycobacterial diadenylate cyclase gene and releases high levels of the STING agonist bis-(3’-5’)-cyclic dimeric adenosine monophosphate (c-di-AMP). We used a 24-week guinea pig vaccination-Mycobacterium tuberculosis (M.tb.) challenge model to test the protective efficacy of BCG-disA-OE versus wild-type BCG and measured lung weights, pathology scores, and M.tb. organ colony-forming unit (CFU) counts. Results BCG-disA-OE elicited significantly stronger tumor necrosis factor-α, interleukin (IL)-6, IL-1β, interferon (IFN) regulatory factor 3, and IFN-β levels than BCG-wild type (WT) in vitro in murine macrophages. In vivo in guinea pigs, we found that BCG-disA-OE reduced lung weights, pathology scores, and M.tb. CFU counts in lungs by 28% (P &lt; .05), 34%, and 2.0 log10 CFU units (P &lt; .05) compared with BCG-WT, respectively. Conclusions We report a strategy of delivering a STING agonist from within live BCG. Overproduction of the STING agonist c-di-AMP significantly enhanced the protective efficacy of BCG against pulmonary and extrapulmonary tuberculosis. Our findings support the development of BCG-vectored STING agonists as a tuberculosis vaccine strategy.

BackgroundIn spite of a consistent protection against tuberculosis (TB) in children, Mycobacterium bovis Bacille Calmette-Guerin (BCG) fails to provide adequate protection against the disease in adults as well as against reactivation of latent infections or exogenous reinfections. It has been speculated that failure to generate adequate memory T cell response, elicitation of inadequate immune response against latency-associated antigens and inability to impart long-term immunity against M. tuberculosis infections are some of the key factors responsible for the limited efficiency of BCG in controlling TB.Methods/Principal FindingsIn this study, we evaluated the ability of a DNA vaccine expressing α-crystallin- a key latency antigen of M. tuberculosis to boost the BCG induced immunity. ‘BCG prime – DNA boost’ regimen (B/D) confers robust protection in guinea pigs along with a reduced pathology in comparison to BCG vaccination (1.37 log10 and 1.96 log10 fewer bacilli in lungs and spleen, respectively; p<0.01). In addition, B/D regimen also confers enhanced protection in mice. Further, we show that B/D immunization in mice results in a heightened frequency of PPD and antigen specific multi-functional CD4 T cells (3+) simultaneously producing interferon (IFN)γ, tumor necrosis factor (TNF)α and interleukin (IL)2.Conclusions/SignificanceThese results clearly indicate the superiority of α-crystallin based B/D regimen over BCG. Our study, also demonstrates that protection against TB is predictable by an increased frequency of 3+ Th1 cells with superior effector functions. We anticipate that this study would significantly contribute towards the development of superior booster vaccines for BCG vaccinated individuals. In addition, this regimen can also be expected to reduce the risk of developing active TB due to reactivation of latent infection.

BackgroundInfectious diseases are the second leading cause of death worldwide. In order to better understand and treat them, an accurate evaluation using multi-modal imaging techniques for anatomical and functional characterizations is needed. For non-invasive imaging techniques such as computed tomography (CT), magnetic resonance imaging (MRI), and positron emission tomography (PET), there have been many engineering improvements that have significantly enhanced the resolution and contrast of the images, but there are still insufficient computational algorithms available for researchers to use when accurately quantifying imaging data from anatomical structures and functional biological processes. Since the development of such tools may potentially translate basic research into the clinic, this study focuses on the development of a quantitative and qualitative image analysis platform that provides a computational radiology perspective for pulmonary infections in small animal models. Specifically, we designed (a) a fast and robust automated and semi-automated image analysis platform and a quantification tool that can facilitate accurate diagnostic measurements of pulmonary lesions as well as volumetric measurements of anatomical structures, and incorporated (b) an image registration pipeline to our proposed framework for volumetric comparison of serial scans. This is an important investigational tool for small animal infectious disease models that can help advance researchers’ understanding of infectious diseases.MethodsWe tested the utility of our proposed methodology by using sequentially acquired CT and PET images of rabbit, ferret, and mouse models with respiratory infections of Mycobacterium tuberculosis (TB), H1N1 flu virus, and an aerosolized respiratory pathogen (necrotic TB) for a total of 92, 44, and 24 scans for the respective studies with half of the scans from CT and the other half from PET. Institutional Administrative Panel on Laboratory Animal Care approvals were obtained prior to conducting this research. First, the proposed computational framework registered PET and CT images to provide spatial correspondences between images. Second, the lungs from the CT scans were segmented using an interactive region growing (IRG) segmentation algorithm with mathematical morphology operations to avoid false positive (FP) uptake in PET images. Finally, we segmented significant radiotracer uptake from the PET images in lung regions determined from CT and computed metabolic volumes of the significant uptake. All segmentation processes were compared with expert radiologists’ delineations (ground truths). Metabolic and gross volume of lesions were automatically computed with the segmentation processes using PET and CT images, and percentage changes in those volumes over time were calculated. (Continued on next page)(Continued from previous page) Standardized uptake value (SUV) analysis from PET images was conducted as a complementary quantitative metric for disease severity assessment. Thus, severity and extent of p...