Evaluation of the cytosol escape of actinobacteria from the phagosome of human macrophages

Abstract

The success of infection by some intracellular pathogens is due to their ability to resist bactericidal components of host cells, such as phagocytosis. This ability to surpass the immune response can be given by different resistance mechanisms, such as the structure of the bacterium cell wall and the use of specific enzymes and toxins, which allow the pathogens to inhabit the intracellular environment and remain in an favorable space to escape the host immune response and antimicrobial drugs. Intracellular pathogens, such as Mycobacterium tuberculosis, after being phagocytized, survive in the intracellular environment because of their ability to inhibit the fusion of lysosomal vesicles with the phagosome and also by the capacity to escape the phagosome, accessing the cytosol. Bacteria that belong to the Mycobacterium tuberculosis Complex (MTBC) are responsible for causing tuberculosis in humans and animals, leading to serious problems in public and animal health. Mycobacterium tuberculosis is the major global cause of mortality from a single infectious agent, with 1.6 million deaths and ten million cases reported due to tuberculosis every year. The efficiency of the mycobacterial evasion mechanisms described above is associated with the composition of its cell wall, consisting of a thick layer of complex lipids, including mycolic acids, and arabinogalactan, and its type VII secretion system. Mycolic acids give mycobacteria important features of resistance to drugs and environmental stress, including dissection and pH extremes. Bacteria that have this lipid complexity in the composition of their cell wall are part of the class of Actinobacteria. One of the goals of the "Jovem Pesquisador" Project in which this scientific initiation proposal is linked to is to evaluate the escape from phagosomes to the cytosol of several MTBC species in the human macrophage. These pathogens are classified as biosafety level 3 organisms, requiring specific biocontainment infrastructure and specialized training. Therefore, we propose the use of another Actinobacterium, called Rhodococcus equi, in the standardization and evaluation of the protocol to detect bacterial escape to the cytosol. Rhodococcus equi is a level 2 pathogen, has veterinary importance and zoonotic potential, and is capable of forming granulomatous or piogranulomatous lesions in foals. This pathogen has a relevant plasticity to adapt and survive within the host, inhibiting the formation of phagolysosome and also resisting the acidic pH of the vacuoles that progress to fusion with the lysosome. Nevertheless, its ability to escape and replicate in the macrophage cytoplasm has not yet been reported. It is not known whether R. equi is able to escape from the phagosome to the cytosol to evade the macrophage bactericidal response and to take advantage of an environment rich in nutrients for replication and later propagation to other cells. Thus, the objective of this work is to standardize the technique based on FRET (fluorescence resonance energy transfer) for the detection of bacterial escape from the phagosome to the cytosol using R. equi as a test model. The results of this study will later be used to evaluate the escape of species from MTBC in a level 3+ biosafety laboratory as part of the "Jovem Pesquisador" Project. (AU)