Bacillus Calmette-Guérin Overexpressing an Endogenous Stimulator of Interferon Genes Agonist Provides Enhanced Protection Against Pulmonary Tuberculosis.

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-1ß, interferon (IFN) regulatory factor 3, and IFN-ß 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 < .05), 34%, and 2.0 log10 CFU units (P < .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.

Inhibition of innate immune cytosolic surveillance by an M. tuberculosis phosphodiesterase.

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.

Label-Free Citrate-Stabilized Silver Nanoparticles-Based, Highly Sensitive, Cost-Effective, and Rapid Visual Method for the Differential Detection of Mycobacterium tuberculosis and Mycobacterium bovis.

Tuberculosis poses a global health challenge, and it demands improved diagnostics and therapies. Distinguishing between Mycobacterium tuberculosis (M. tb) and Mycobacterium bovis (M. bovis) infections holds critical "One Health" significance due to the zoonotic nature of these infections and inherent resistance of M. bovis to pyrazinamide, a key part of the directly observed treatment, short-course (DOTS) regimen. Furthermore, most of the currently used molecular detection methods fail to distinguish between the two species. To address this, our study presents an innovative molecular-biosensing strategy. We developed a label-free citrate-stabilized silver nanoparticle aggregation assay that offers sensitive, cost-effective, and swift detection. For molecular detection, genomic markers unique to M. tb and M. bovis were targeted using species-specific primers. In addition to amplifying species-specific regions, these primers also aid the detection of characteristic deletions in each of the mycobacterial species. Post polymerase chain reaction (PCR), we compared two highly sensitive visual detection methods with respect to the traditional agarose gel electrophoresis. The paramagnetic bead-based bridging flocculation assay successfully discriminates M. tb from M. bovis with a sensitivity of ∼40 bacilli. The second strategy exploits citrate-stabilized silver nanoparticles, which aggregate in the absence of amplified dsDNA on the addition of sodium chloride (NaCl). This technique enables the precise, sensitive, and differential detection of as few as ∼4 bacilli. Our study hence advances tuberculosis detection, overcoming the challenges of M. tb and M. bovis differentiation and offering a quicker alternative to time-consuming methods.

Isothermal recombinase polymerase amplification and silver nanoparticle assay: a sustainable approach for ultrasensitive detection of Klebsiella pneumoniae.

Our study addresses the urgent need for effective detection of Klebsiella pneumoniae, a recognized threat by the World Health Organization (WHO). Current challenges in managing K. pneumoniae infections include the lack of rapid and affordable detection tools, particularly in resource-limited point-of-care (POC) settings. To tackle this, we developed an innovative molecular detection pipeline combining three POC-compatible methods. Firstly, we employed Insta DNA™ card-based sample collection and DNA extraction for simplicity and ease of use. Next, we utilized recombinase polymerase amplification (RPA) targeting the Klebsiella hemolysin gene, khe, specific to the K. pneumoniae species complex (KpSC). Finally, we integrated a silver nanoparticle (AgNP) aggregation assay for visual detection, offering a rapid, sensitive, and specific method capable of detecting as few as ∼3 bacteria of K. pneumoniae within ∼45 minutes. This approach eliminates the need for complex equipment, making it highly suitable for field and resource-limited POC applications. Moreover, our method introduces an environmentally significant detection strategy. The method developed minimizes chemical reagent usage and reduces the carbon footprint associated with sample transportation. Furthermore, our method reduces waste compared to the traditional detection techniques, offering a safer alternative to ethidium bromide or other DNA dyes which are often genotoxic and mutagenic in nature. Silver nanoparticles, being environmentally safer, can also be recycled from the waste, contributing to sustainability in nanoparticle production and disposal. Overall, our technique presents a promising solution for detecting K. pneumoniae in various settings, including environmental, water, and food samples, as well as industrial or hospital effluents. By aligning with global efforts to improve public health and environmental sustainability, our approach holds significant potential for enhancing disease management and reducing environmental impact.

Discovery of potent antimycobacterial agents targeting lumazine synthase (RibH) of Mycobacterium tuberculosis.

Tuberculosis (TB) continues to be a global health crisis, necessitating urgent interventions to address drug resistance and improve treatment efficacy. In this study, we validate lumazine synthase (RibH), a vital enzyme in the riboflavin biosynthetic pathway, as a potential drug target against Mycobacterium tuberculosis (M. tb) using a CRISPRi-based conditional gene knockdown strategy. We employ a high-throughput molecular docking approach to screen ~ 600,000 compounds targeting RibH. Through in vitro screening of 55 shortlisted compounds, we discover 3 compounds that exhibit potent antimycobacterial activity. These compounds also reduce intracellular burden of M. tb during macrophage infection and prevent the resuscitation of the nutrient-starved persister bacteria. Moreover, these three compounds enhance the bactericidal effect of first-line anti-TB drugs, isoniazid and rifampicin. Corroborating with the in silico predicted high docking scores along with favourable ADME and toxicity profiles, all three compounds demonstrate binding affinity towards purified lumazine synthase enzyme in vitro, in addition these compounds exhibit riboflavin displacement in an in vitro assay with purified lumazine synthase indicative of specificity of these compounds to the active site. Further, treatment of M. tb with these compounds indicate reduced production of flavin adenine dinucleotide (FAD), the ultimate end product of the riboflavin biosynthetic pathway suggesting the action of these drugs on riboflavin biosynthesis. These compounds also show acceptable safety profile in mammalian cells, with a high selective index. Hence, our study validates RibH as an important drug target against M. tb and identifies potent antimycobacterial agents.

Multidimensional futuristic approaches to address the pandemics beyond COVID-19.

Globally, the impact of the coronavirus disease 2019 (COVID-19) pandemic has been enormous and unrelenting with ∼6.9 million deaths and ∼765 million infections. This review mainly focuses on the recent advances and potentially novel molecular tools for viral diagnostics and therapeutics with far-reaching implications in managing the future pandemics. In addition to briefly highlighting the existing and recent methods of viral diagnostics, we propose a couple of potentially novel non-PCR-based methods for rapid, cost-effective, and single-step detection of nucleic acids of viruses using RNA mimics of green fluorescent protein (GFP) and nuclease-based approaches. We also highlight key innovations in miniaturized Lab-on-Chip (LoC) devices, which in combination with cyber-physical systems, could serve as ideal futuristic platforms for viral diagnosis and disease management. We also discuss underexplored and underutilized antiviral strategies, including ribozyme-mediated RNA-cleaving tools for targeting viral RNA, and recent advances in plant-based platforms for rapid, low-cost, and large-scale production and oral delivery of antiviral agents/vaccines. Lastly, we propose repurposing of the existing vaccines for newer applications with a major emphasis on Bacillus Calmette-Guérin (BCG)-based vaccine engineering.

Augmentation of the Riboflavin-Biosynthetic Pathway Enhances Mucosa-Associated Invariant T (MAIT) Cell Activation and Diminishes Mycobacterium tuberculosis Virulence.

Mucosa-associated invariant T (MAIT) cells play a critical role in antimicrobial defense. Despite increased understanding of their mycobacterial ligands and the clinical association of MAIT cells with tuberculosis (TB), their function in protection against Mycobacterium tuberculosis infection remains unclear. Here, we show that overexpressing key genes of the riboflavin-biosynthetic pathway potentiates MAIT cell activation and results in attenuation of M. tuberculosis virulence in vivo. Further, we observed greater control of M. tuberculosis infection in MAIThi CAST/EiJ mice than in MAITlo C57BL/6J mice, highlighting the protective role of MAIT cells against TB. We also endogenously adjuvanted Mycobacterium bovis BCG with MR1 ligands via overexpression of the lumazine synthase gene ribH and evaluated its protective efficacy in the mouse model of M. tuberculosis infection. Altogether, our findings demonstrate that MAIT cells confer host protection against TB and that overexpression of genes in the riboflavin-biosynthetic pathway attenuates M. tuberculosis virulence. Enhancing MAIT cell-mediated immunity may also offer a novel approach toward improved vaccines against TB. IMPORTANCE Mucosa-associated invariant T (MAIT) cells are an important subset of innate lymphocytes that recognize microbial ligands derived from the riboflavin biosynthesis pathway and mediate antimicrobial immune responses. Modulated MAIT cell responses have been noted in different forms of tuberculosis. However, it has been unclear if increased MAIT cell abundance is protective against TB disease. In this study, we show that augmentation of the mycobacterial MAIT cell ligands leads to higher MAIT cell activation with reduced M. tuberculosis virulence and that elevated MAIT cell abundance confers greater control of M. tuberculosis infection. Our study also highlights the potential of endogenously adjuvanting the traditional BCG vaccine with MR1 ligands to augment MAIT cell activation. This study increases current knowledge on the roles of the riboflavin-biosynthetic pathway and MAIT cell activation in M. tuberculosis virulence and host immunity against TB.

A bacterial cyclic dinucleotide activates the cytosolic surveillance pathway and mediates innate resistance to tuberculosis.

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 (IFN) responses, which are crucial to antimicrobial defense. However, the mechanisms and role of c-di-AMP signaling in Mycobacterium tuberculosis virulence remain unclear. Here we show that resistance to tuberculosis 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)-overexpressing M. tuberculosis strain that secretes excess c-di-AMP activates the interferon regulatory factor (IRF) pathway with enhanced levels of IFN-ß, elicits increased macrophage autophagy, and exhibits substantial virulence attenuation in mice. We show that c-di-AMP-mediated IFN-ß induction during M. tuberculosis infection requires stimulator of interferon genes (STING)-signaling. We observed that c-di-AMP induction of IFN-ß is independent of the cytosolic nucleic acid receptor cyclic GMP-AMP (cGAMP) synthase (cGAS), but cGAS nevertheless contributes substantially to the overall IFN-ß 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 tuberculosis.