The host perspective: understanding phagosome biology and immune control of phagosome maturation.

The aim of our research is to give insights into the host factors that facilitate mycobacterial killing. The exact mechanisms that govern this initial sterilizing reaction are not completely understood. Understanding therefore how host cells eliminate mycobacteria is important to find possible therapeutic strategies that enhance this natural response.
The phagosome is a key organelle that links innate and adaptive immunity. The phagosome that contains mycobacteria is a fascinating compartment customized by the bacteria for its own benefit. Understanding how the circuits between mycobacteria and phagosomal milieu signals are established is crucial. Not only because in some cases this response is effective but also because in case of persistence, the mycobacteria/phagosome interface must be maintained trough time.
We have identified a large number of membrane trafficking regulatory proteins regulated at the transcriptional level via NF-kB during the early killing of mycobacteria by macrophages. This argues that NF-kB participates in the early steps of the innate immune response altering the levels of proteins that regulates phagolysosome biogenesis (Gutierrez et al. 2008; Gutierrez et al. 2009). The most promising candidate proteins are membrane trafficking proteins and lysosomal receptors. However, the exact role of many of these proteins in phagosome maturation and innate immunity is unknown. We aim to define the function of these proteins during phagocytosis and evaluate the impact on the innate immune response to M. tuberculosis. For one of the identified proteins, sortilin, we have already shown its role during phagosome maturation. We identified a novel pathway by which selected proteins are transported to the phagosome bypassing lysosomes during maturation of phagosomes (Wähe et al. 2010).
We expect that the knowledge emerging from our studies will identify critical host anti-mycobacterial factors. The characterization of those factors will highlight attractive targets subverted by intracellular mycobacteria to develop new strategies to combat tuberculosis.

The M. tuberculosis perspective: understanding adaptation to the phagosome environment.

Mycobacterial persistence is linked to two of the major obstacles against the eradication of tuberculosis: a large reservoir of people without clinical symptoms and complications in the treatment with the majority of antibiotics. In spite of its enormous relevance and clinical implications, the fundamental biology and a mechanistic understanding of mycobacterial persistence remains a puzzle for biologists.
We are currently establishing a model of persistence that considers one of the most important aspects of mycobacterial lifestyle: intracellularity. In our group, we are developing, characterizing and validating this model as a new dynamic system to address questions in the field of mycobacterial persistence. For that, we combine live cell imaging and phenotypic analysis to provide insights into the mechanism of persistence. We also aim in the future to exploit this cellular system as a validated and predictive platform to find new effective chemical compounds against intracellularly persistent mycobacteria.
By using whole-genome sequencing and bioinformatics analysis, we are investigating the genetic modifications that are associated to intracellular persistent mycobacteria. We also analyze the phenotype that made these bacteria persist in host cells. The expression profile of the intracellular persisters will provide important and new components that are critical for mycobacterial persistence in a cell host context. We undertake this question performing microarray gene expression of the mycobacterial persisters. The gene expression profile comparison with non persistent mycobacteria will contribute to identify genes expressed during persistence. Combining this information with the genome sequence of the persisters we will be able to provide an integrative analysis of the factors that make mycobacteria persist in host cells.

Our ultimate goal is to understand how the long-term manipulation of the phagosomal fate leads to mycobacterial persistence. Our findings will give important insights into the intracellular mechanism of mycobacterial persistence and evolution of the interaction between mycobacteria and host cells. Our goal is to translate these concepts to more complex (and still imperfect) animal models to develop new therapies against latent infection.