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Mycobacterium tuberculosis is able to colonize, persist, and massively replicate in host cells, such as phagocytes and epithelial cells. The intracellular stage of the bacteria is critical to the development of tuberculosis pathogenesis. The detailed mechanisms of intracellular trafficking of the bacillus are not fully understood and require further investigations. Therefore, increasing the knowledge of this process will help to develop therapeutic tools that will lower the burden of tuberculosis. M. tuberculosis is genetically tractable and tolerates the expression of heterologous fluorescent proteins. Thus, the intracellular distribution of the bacteria expressing fluorescent tracers can be easily defined using confocal microscopy. Advances in imaging techniques and images-based analysis allow the rapid quantification of biological objects in complex environments. In this chapter, we detailed high-content / high-throughput imaging methods to track the bacillus within host cell settings.

Mycobacterium tuberculosis is able to colonize, persist, and massively replicate in host cells, such as phagocytes and epithelial cells. The intracellular stage of the bacteria is critical to the development of tuberculosis pathogenesis. The detailed mechanisms of intracellular trafficking of the bacillus are not fully understood and require further investigations. Therefore, increasing the knowledge of this process will help to develop therapeutic tools that will lower the burden of tuberculosis. M. tuberculosis is genetically tractable and tolerates the expression of heterologous fluorescent proteins. Thus, the intracellular distribution of the bacteria expressing fluorescent tracers can be easily defined using confocal microscopy. Advances in imaging techniques and images-based analysis allow the rapid quantification of biological objects in complex environments. In this chapter, we detailed high-content / high-throughput imaging methods to track the bacillus within host cell settings.

Mycobacterium tuberculosis (Mtb), the pathogen causing human tuberculosis, has evolved multiple strategies to successfully prevent clearance by immune cells and to establish dissemination and long-term survival in the host. The modulation of host immunity to maximize pathogen elimination while minimizing inflammation-mediated tissue damage may provide another tool to fight drug-resistant Mtb strains. Metabolic reprogramming of immune cell populations can dramatically influence the outcome of immune responses and modulate antimicrobial properties of infected host cells, nicely demonstrating that metabolites are tightly linked to immune cell effector functions. One important endogenous metabolite of the Krebs cycle is itaconate, which has potent bactericidal activity by inhibiting isocitrate lyase and the glyoxylate shunt within prokaryotes including mycobacteria. Recent findings show that itaconate and the catalytic enzyme responsible for its generation in mammalian cells, i.e. IRG1 (immune-responsive gene 1), also modify inflammatory signaling of infected cells enhancing host defense pathways. Here, we demonstrate that IRG1 is recruited to Mtb-containing phagosomes and that it influences the host response controlling Mtb infection. While IRG1 deficiency does not affect uptake of Mtb by macrophages and dendritic cells (DCs) in vitro, it increases the intracellular replication of Mtb. Concomitantly, in comparison to wild type cells, IRG1-deficient macrophages and DCs have increased levels of lipid droplets, a correlate of inflammation. These intracellular organelles store triacylglycerol and phospholipids that are hijacked by Mtb as reservoir of host nutrients. Exposure of IRG1-deficient mice to M. bovis BCG via the intranasal route induced neither lethality nor severe lung immunopathology, while IRG1-deficient mice were highly susceptible to Mtb infection resulting in animal death three weeks post-infection linked to exacerbated inflammation and high mycobacterial burden. The lungs of infected IRG1-deficient mice displayed large areas of necrotizing granulomatous inflammation and neutrophil infiltration, accompanied by reduced levels of B and T lymphocytes and increased levels of alveolar and interstitial macrophage populations, compared to their wild type counterparts. Therefore, our findings demonstrate that IRG1 is a major player in controlling the acute phase of Mtb infection with a specific effect on pathogenic mycobacteria.

Multi-drug-resistant tuberculosis (TB) is a major public health problem, concerning about half a million cases each year. Patients hardly adhere to the current strict treatment consisting of more than 10 000 tablets over a 2-year period. There is a clear need for efficient and better formulated medications. We have previously shown that nanoparticles made of cross-linked poly-β-cyclodextrins (pβCD) are efficient vehicles for pulmonary delivery of powerful combinations of anti-TB drugs. Here, we report that in addition to being efficient drug carriers, pβCD nanoparticles are endowed with intrinsic antibacterial properties. Empty pβCD nanoparticles are able to impair Mycobacterium tuberculosis (Mtb) establishment after pulmonary administration in mice. pβCD hamper colonization of macrophages by Mtb by interfering with lipid rafts, without inducing toxicity. Moreover, pβCD provoke macrophage apoptosis, leading to depletion of infected cells, thus creating a lung microenvironment detrimental to Mtb persistence. Taken together, our results suggest that pβCD nanoparticles loaded or not with antibiotics have an antibacterial action on their own and could be used as a carrier in drug regimen formulations effective against TB., SCOPUS: ar.j, info:eu-repo/semantics/published