A critical feature of Mycobacterium tuberculosis, the causative agent of human tuberculosis (TB), is its ability to survive and multiply within macrophages, making these host cells an ideal niche for persisting microbes. Killing the intracellular tubercle bacilli is a key requirement for efficient tuberculosis treatment, yet identifying potent inhibitors has been hampered by labor-intensive techniques and lack of validated targets. Here, we present the development of a phenotypic cell-based assay that uses automated confocal fluorescence microscopy for high throughput screening of chemicals that interfere with the replication of M. tuberculosis within macrophages. Screening a library of 57,000 small molecules led to the identification of 135 active compounds with potent intracellular anti-mycobacterial efficacy and no host cell toxicity. Among these, the dinitrobenzamide derivatives (DNB) showed high activity against M. tuberculosis, including extensively drug resistant (XDR) strains. More importantly, we demonstrate that incubation of M. tuberculosis with DNB inhibited the formation of both lipoarabinomannan and arabinogalactan, attributable to the inhibition of decaprenyl-phospho-arabinose synthesis catalyzed by the decaprenyl-phosphoribose 2′ epimerase DprE1/DprE2. Inhibition of this new target will likely contribute to new therapeutic solutions against emerging XDR-TB. Beyond validating the high throughput/content screening approach, our results open new avenues for finding the next generation of antimicrobials.
Vaccination against tumor antigens has been shown to be a safe and efficacious prophylactic and therapeutic antitumor treatment in many animal models. Clinical studies in humans indicate that specific immunotherapy can also result in clinical benefits. The active pharmaceutical ingredient in such vaccines can be DNA, RNA, protein, or peptide and can be administered naked, encapsulated, or after delivery in vitro into cells that are then adoptively transferred. One of the easiest, most versatile and theoretically safest technologies relies on the direct injection of naked messenger RNA (mRNA) that code for tumor antigens. We and others have shown in mice that intradermal application of naked mRNA results in protein expression and the development of an immune response. We used this protocol to vaccinate 15 melanoma patients. For each patient a growing metastasis was removed, total RNA was extracted, reverse-transcribed, amplified, and cloned. Libraries of cDNA were transcribed to produce unlimited amounts of copy mRNA. Autologous preparations were applied intradermally in combination with granulocyte macrophage colony-stimulating factor as adjuvant. We demonstrate here that such treatment is feasible and safe (phase 1 criteria). Furthermore, an increase in antitumor humoral immune response was seen in some patients. However, a demonstration of clinical effectiveness of direct injection of copy mRNA for antitumor immunotherapy was not shown in this study and must be evaluated in subsequent trials.
We reported that RNA condensed on protamine is protected from RNase-mediated degradation and can be used for vaccination. Here, we show that such complexes are also danger signals that activate mouse cells through a MyD88-dependent pathway. Moreover, mRNA-protamine complexes stimulate human blood cells. They strongly activate DC and monocytes, leading to TNF-a and IFN-a secretion. In addition, protamine-RNA complexes directly activate B cells, NK cells and granulocytes. The detailed analysis of the activated cell types, the study of the cytokines released from PBMC cultured with protamine-RNA complexes and recently published results suggest that TLR-7 and TLR-8 may be involved in the recognition of protamine-stabilized RNA. Our data indicate that protamine-stabilized RNA, which may be similar to RNA condensed in the nucleocapsids of RNA viruses, is a strong danger signal. Thus, similarly to plasmid DNA, protamine-RNA combines antigen production and non-specific immunostimulation. The studies presented here explain the capacity of protamine-RNA to act as a vaccine, and pave the way towards the development of safe and efficient mRNA-based immunotherapies.
The ability of the tubercle bacillus to arrest phagosome maturation is considered one major mechanism that allows its survival within host macrophages. To identify mycobacterial genes involved in this process, we developed a high throughput phenotypic cell-based assay enabling individual sub-cellular analysis of over 11,000 Mycobacterium tuberculosis mutants. This very stringent assay makes use of fluorescent staining for intracellular acidic compartments, and automated confocal microscopy to quantitatively determine the intracellular localization of M. tuberculosis. We characterised the ten mutants that traffic most frequently into acidified compartments early after phagocytosis, suggesting that they had lost their ability to arrest phagosomal maturation. Molecular analysis of these mutants revealed mainly disruptions in genes involved in cell envelope biogenesis (fadD28), the ESX-1 secretion system (espL/Rv3880), molybdopterin biosynthesis (moaC1 and moaD1), as well as in genes from a novel locus, Rv1503c-Rv1506c. Most interestingly, the mutants in Rv1503c and Rv1506c were perturbed in the biosynthesis of acyltrehalose-containing glycolipids. Our results suggest that such glycolipids indeed play a critical role in the early intracellular fate of the tubercle bacillus. The unbiased approach developed here can be easily adapted for functional genomics study of intracellular pathogens, together with focused discovery of new anti-microbials.
Since direct injection of naked mRNA induces an immune response, we tested the capacity of RNA to signal danger. We show here that mRNA molecules that are protected from immediate degradation either through interaction with cationic proteins (trans protection) or through chemical modification of the phosphodiester backbone (phosphorothioate RNA; cis protection) act as sequence-independent danger signals on mouse DC. As opposed to CpG DNA, the cis-stabilized RNA is degraded in a few minutes, does not activate B cells and, in contrast to double-stranded RNA, requires MyD88 for activation of the DC. We postulate that phosphorothioate RNA, which mimics trans-stabilized RNA, is a new PAMP.
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