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.

Immunomodulating microRNAs of mycobacterial infections

“…Actin is a key player in the phagocytic process and its dynamics are involved in the motility of the cell, in the formation of pseudopodia for the internalization of extracellular particles and in vesicle trafficking [36]. For example, our results have indicated a role for miR-142-3p in controlling actin dynamics during infection with implications on bacteria internalization by macrophages [37]. miR-142-3p regulates N-WASP [37], a signal transducer for cell surface receptors leading to actin assembly via the Arp2/3 complex [36].…”

“…For example, our results have indicated a role for miR-142-3p in controlling actin dynamics during infection with implications on bacteria internalization by macrophages [37]. miR-142-3p regulates N-WASP [37], a signal transducer for cell surface receptors leading to actin assembly via the Arp2/3 complex [36]. Furthermore, mycobacteria promote the over-expression of this miRNA at early times of phagocytosis in macrophages.…”

Acting on Actin During Bacterial Infection

“…An important class of Mycobacterium pathogens includes tuberculosis bacilli. This intracellular facultative pathogen controls the bacterial load during macrophage internalization by interfering with actin polymerization at the phagocytic cup [21]. This is a necessary step in virulence for preventing apoptosis and therefore to prevent pathogen intracellular killing [22].…”

“…This is a necessary step in virulence for preventing apoptosis and therefore to prevent pathogen intracellular killing [22]. For this, during early phases of Mycobacterium infection, the microRNA 142-3p is overexpressed in response to phagocytosis and interferes with the expression of N-WASP and consequently with the Arp2/3 complex required for actin nucleation at the cell membrane [21]. Therefore, a low bacterial load is accomplished intracellularly, preventing the apoptosis of the infected cells.…”

“…During M. tuberculosis as well as for M. marinum infection phagolysosomal rupture and bacteria escape to the cytosol usually leads to necrotic cell death [61,62]. The existence of a functional RD1 region expressing ESAT-6 is relevant for the activation of the inflammasome, the necrotic cell death and the secretion of proinflammatory cytokines IL-1β [21]. In endothelial cells, however, the tubercle bacilli survives [47].…”