title: Actin-binding protein regulation by microRNAs as a novel microbial strategy to modulate phagocytosis by host cells: the case of N-Wasp and miR-142-3p.
abstract: Mycobacterium tuberculosis (Mtb) is a successful intracellular pathogen that thrives in macrophages (Mφs). There is a need to better understand how Mtb alters cellular processes like phagolysosome biogenesis, a classical determinant of its pathogenesis. A central feature of this bacteria's strategy is the manipulation of Mφ actin. Here, we examined the role of microRNAs (miRNAs) as a potential mechanism in the regulation of actin-mediated events leading to phagocytosis in the context of mycobacteria infection. Given that non-virulent Mycobacterium smegmatis also controls actin filament assembly to prolong its intracellular survival inside host cells, we performed a global transcriptomic analysis to assess the modulation of miRNAs upon M. smegmatis infection of the murine Mφ cell line, J774A.1. This approach identified miR-142-3p as a key candidate to be involved in the regulation of actin dynamics required in phagocytosis. We unequivocally demonstrate that miR-142-3p targets N-Wasp, an actin-binding protein required during microbial challenge. A gain-of-function approach for miR-142-3p revealed a down-regulation of N-Wasp expression accompanied by a decrease of mycobacteria intake, while a loss-of-function approach yielded the reciprocal increase of the phagocytosis process. Equally important, we show Mtb induces the early expression of miR-142-3p and partially down-regulates N-Wasp protein levels in both the murine J774A.1 cell line and primary human Mφs. As proof of principle, the partial siRNA-mediated knock down of N-Wasp resulted in a decrease of Mtb intake by human Mφs, reflected in lower levels of colony-forming units (CFU) counts over time. We therefore propose the modulation of miRNAs as a novel strategy in mycobacterial infection to control factors involved in actin filament assembly and other early events of phagolysosome biogenesis.
authors.affiliation: Centro de Patogénese Molecular, Faculdade de Farmácia, Unidade dos Retrovírus e Infecções Associadas e Instituto de Medicina Molecular, Universidade de Lisboa Lisboa, Portugal.
authors.forename: Paulo
authors.initials: P
authors.lastname: Bettencourt
authors.forename: Sabrina
authors.initials: S
authors.lastname: Marion
authors.forename: David
authors.initials: D
authors.lastname: Pires
authors.forename: Leonor F
authors.initials: LF
authors.lastname: Santos
authors.forename: Claire
authors.initials: C
authors.lastname: Lastrucci
authors.forename: Nuno
authors.initials: N
authors.lastname: Carmo
authors.forename: Jonathon
authors.initials: J
authors.lastname: Blake
authors.forename: Vladimir
authors.initials: V
authors.lastname: Benes
authors.forename: Gareth
authors.initials: G
authors.lastname: Griffiths
authors.forename: Olivier
authors.initials: O
authors.lastname: Neyrolles
authors.forename: Geanncarlo
authors.initials: G
authors.lastname: Lugo-Villarino
authors.forename: Elsa
authors.initials: E
authors.lastname: Anes
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D020869:Gene Expression Profiling; D054884:Host-Pathogen Interactions; D006801:Humans; D051379:Mice; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D020102:Mycobacterium smegmatis / Q000276:immunology / Q000472:pathogenicity; D009169:Mycobacterium tuberculosis / Q000276:immunology* / Q000472:pathogenicity; D010587:Phagocytosis; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal / Q000235:genetics / Q000378:metabolism*
chemical_list: C555159:MIRN142 microRNA, human; D035683:MicroRNAs; C529102:Mirn142 microRNA, mouse; C495771:WASL protein, human; C495772:Wasl protein, mouse; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal
keywords: M. smegmatis; M. tuberculosis; N-Wasp; macrophage; miR-142-3p; miRNA; phagocytosis; tuberculosis