Characterization of novel double-reporter strains of Mycobacterium abscessus for drug discovery: a study in mScarlet.

Mycobacterium abscessus (Mab) is an emerging pathogen that poses a severe health threat, especially in people with cystic fibrosis and other chronic lung diseases. Available drugs are largely ineffective due to an exquisite intrinsic resistance, making Mab infections only comparable to multidrug-resistant tuberculosis. Current treatment is based on lengthy multidrug therapy, complicated by poor outcomes and high rates of treatment failure, recurrence, and mortality. Thus, finding new and more efficient drugs to combat this pathogen is urgent. However, drug discovery efforts targeting Mab have been limited, and traditional drug screening methods are labor-intensive, low-throughput, and do not reflect clinical effectiveness. Therefore, this work aimed to develop a new, efficient, and reliable tool for drug screening against Mab that can be used in vitro for identifying hits in a high-throughput manner and in vivo to select drug candidates for future clinical trials. We engineered two stable double-reporter strains of Mab capable of emitting strong fluorescent and luminescent signals. This is due to the expression of mScarlet protein and luciferase enzyme or the entire lux operon. Importantly, these strains maintain the same ground characteristics as the non-transformed Mab strain. We show that these new strains can be applied to various setups, from MIC determination in broth cultures and macrophage infection assays to in vivo infection (using the Galleria mellonella model). Using these strains enhances the potential for high-throughput screening of thousands of compounds in a fast and reliable way. IMPORTANCE: Mycobacterium abscessus (Mab) is currently considered an "incurable nightmare." Its intrinsic resistance, high toxicity, long duration, and low cure rates of available therapies often lead to the clinical decision not to treat. Moreover, one of the significant drawbacks of anti-Mab drug development is the lack of correlation between in vitro susceptibility and clinical efficacy. Most drug screening assays are performed on Mab growing in liquid cultures. But being an intracellular pathogen, inducing granulomas and biofilm formation, the broth culture is far from ideal as in vitro drug-testing setup. This study presents new double-reporter Mab strains that allow direct real-time bacterial detection and quantification in a non-invasive way. These strains can be applied to an extensive range of experimental settings, far surpassing the utility of single-reporter bacteria. They can be used in all steps of the pre-clinical anti-Mab drug development pipeline, constituting a highly valuable tool to increase its success.

Mycobacteria-induced anaemia revisited: a molecular approach reveals the involvement of NRAMP1 and lipocalin-2, but not of hepcidin.

Anaemia is a frequent complication of chronic infectious diseases but the exact mechanisms by which it develops remain to be clarified. In the present work, we used a mouse model of mycobacterial infection to study molecular alterations of iron metabolism induced by infection. We show that four weeks after infection with Mycobacterium avium BALB/c mice exhibited a moderate anaemia, which was not accompanied by an increase on hepatic hepcidin mRNA expression. Instead, infected mice presented increased mRNA expression of ferroportin (Slc40a1), ceruloplasmin (Cp), hemopexin (Hpx), heme-oxygenase-1 (Hmox1) and lipocalin-2 (Lcn2). Both the anaemia and the mRNA expression changes of iron-related genes were largely absent in C.D2 mice which bear a functional allele of the Nramp1 gene. Data presented in this work suggest that anaemia due to a chronic mycobacterial infection may develop in the absence of elevated hepcidin expression, is influenced by Nramp1 and may involve lipocalin-2.

Engagement of Toll-like receptor 2 in mouse macrophages infected with Mycobacterium avium induces non-oxidative and TNF-independent anti-mycobacterial activity.

Toll-like receptor (TLR) 2 plays an important role in the immune response to mycobacterial infections, being required for optimal immunity against certain virulent Mycobacterium avium strains. Here we analyzed the role of TLR2 in the intra-macrophagic growth of M. avium, using macrophages from TLR2-deficient mice. We found that the engagement of TLR2/TLR6 and/or TLR2/TLR1 receptors induced bacteriostasis of M. avium inside bone marrow-derived macrophages in a MyD88-dependent way. Additionally, lipoproteins from the cell envelope of M. avium with a molecular mass of 20-25 kDa triggered this TLR2 pathway, leading to a decrease in the growth of the mycobacteria. Although TLR2 engagement induced the production of TNF, this cytokine as well as nitric oxide and superoxide molecules were not necessary for TLR2-mediated bacteriostasis. Finally, TLR ligation did not induce the expression of the 47-kDa guanosine triphosphatase (LRG-47) but it promoted an increased maturation of the phagosome with regards to acquisition of LAMP1. Our data show that triggering TLR2 inhibited M. avium growth by an as-yet-unknown mechanism that may involve increased phagosome maturation.

CD40 is required for the optimal induction of protective immunity to Mycobacterium avium.

C57Bl/6 mice and mice deficient in the CD40 molecule were infected with three strains of Mycobacterium avium. Two of the M. avium strains proliferated more extensively in CD40-deficient (CD40-/-) mice than in control mice. The increased susceptibility to infection of CD40-/- mice was associated with the generation of poorer interleukin-12 (IL-12) p40 and interferon-gamma (IFN-gamma) responses as compared to the controls, suggesting a role for CD40 in the development of protective immunity. In contrast, direct triggering of CD40 on infected macrophages failed to induce any anti-mycobacterial activity in infected macrophages.

Limited role of the Toll-like receptor-2 in resistance to Mycobacterium avium.

The role of the Toll-like receptor (TLR)-2 in the generation of protective immunity to Mycobacterium avium was evaluated using gene-disrupted mice. TLR-2-/- mice were more susceptible than wild-type C57Bl/6 mice to M. avium strains that were able to proliferate in vivo before the development of protective immunity and mycobacteriostasis. In contrast, the elimination of non-virulent strains was not affected by the mutation. The generation of interferon-gamma (IFN-gamma)-producing T cells and the expression of the interleukin-12 p40 gene were reduced in TLR-2-deficient mice as compared to C57Bl/6 mice early during infection with M. avium strain 2447. The generation of protective CD4+ T cells was also compromised in the mutated mice as compared with the controls. Our data show that TLR-2 is required for optimal immunity against certain virulent M. avium strains.

NRAMP1- or cytokine-induced bacteriostasis of Mycobacterium avium by mouse macrophages is independent of the respiratory burst.

Restriction of the growth of Mycobacterium avium was studied in wild-type and p47(phox)-deficient macrophages. The ability of gamma interferon and tumour necrosis factor alpha to induce antimycobacterial activity in bone-marrow-derived macrophages or the expression of the NRAMP1-mediated resistance to M. avium were not affected by the deficiency in p47(phox). The addition of exogenous iron increased mycobacterial growth in macrophages expressing a functional NRAMP1 protein or a mutant NRAMP1 protein. Reactive oxygen species are therefore not involved in the constitutive or induced anti-M. avium activities of the mouse macrophage.