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Abstract
Main survival mechanism of pathogenic mycobacteria is to escape inimical phagolysosomal environment inside the macrophages. Many efforts have been made to unravel the molecular mechanisms behind this process. However, little is known about the involvement of microRNAs (miRNAs) in the regulation of phagolysosomal biosynthesis and maturation. Based on a bottom up approach, we searched for miRNAs that were involved in phagolysosomal processing events in the course of mycobacterial infection of macrophages. After infecting THP-1 derived macrophages with viable and heat killed Mycobacterium bovis BCG (BCG), early time points were identified after co-localization studies of the phagosomal marker protein LAMP1 and BCG. Differences in LAMP1 localization on the phagosomes of both groups were observed at 30 min and 4 h. After in silico based pre-selection of miRNAs, expression analysis at the identified time points revealed down-regulation of three miRNAs: miR-3619-5p, miR-637, and miR-324-3p. Consequently, most likely targets were predicted that were supposed to be mutually regulated by these three studied miRNAs. The lysosomal cysteine protease Cathepsin S (CTSS) and Rab11 family-interacting protein 4 (RAB11FIP4) were up-regulated and were considered to be connected to lysosomal trafficking and autophagy. Interaction studies verified the regulation of CTSS by miR-3619-5p. Down-regulation of CTSS by ectopic miR-3619-5p as well as its specific knockdown by siRNA affected the process of autophagy in THP-1 derived macrophages.
Highlights
Mycobacteria consist of both highly pathogenic species e.g., Mycobacterium tuberculosis and opportunist species such as M. avium
The present study mainly focused on an approach to find miRNAs in phagolysosomal maturation during infection of THP-1 derived macrophages with viable and heat killed (HK) BCG and provided functional aspects to the regulated RNAs
At 30 min and 4 h, LAMP1 signal was visible in HK BCG infection co-localizing with BCG and no equivalent signal was seen in viable BCG infection at 30 min and 4 h
Summary
Mycobacteria consist of both highly pathogenic species e.g., Mycobacterium tuberculosis and opportunist species such as M. avium. The phagosome acquires the proteins Rab and EEA on its membrane during early maturation stages, whereas LAMP1 and Rab are present on late maturation stages (Gorvel et al, 1991; Vieira et al, 2003; Flannagan et al, 2009). To understand mycobacterial survival strategies, a number of different proteins (e.g., Rab, Rab, EEA1, hVPS34 etc.) that are involved in phagosomal maturation have been studied on the surface of mycobacteria containing phagosomes (Gorvel et al, 1991; Christoforidis et al, 1999; Bucci et al, 2000; Fratti et al, 2001; Vieira et al, 2003). There are still gaps in understanding how mycobacteria interfere with endosomal trafficking and its regulation
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