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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.

Bettencourt P, Marion S, Pires D, Santos LF, Lastrucci C, Carmo N, Blake J, Benes V, Griffiths G, Neyrolles O, Lugo-Villarino G, Anes E - Front Cell Infect Microbiol (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Centro de Patogénese Molecular, Faculdade de Farmácia, Unidade dos Retrovírus e Infecções Associadas e Instituto de Medicina Molecular, Universidade de Lisboa Lisboa, Portugal.

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.

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N-Wasp is a target of miR-142-3p. (A) Luciferase assay showing the specific targeting of the 3′UTR of the mRNA of Wasl by the miR-142-3p. Data are represented as the mean fold change per sample ± SD (*P ≤ 0.01). (B) Relative expression of miR-142-3p in J774A.1 macrophages infected with M. smegmatis or M. tuberculosis (MOI 10), as measured by the EXIQON (DK) microRNA qPCR services. Data is represented as the mean fold change per sample ± SD at 1, 4, and 24 h post-infection (*P ≤ 0.05 relative to control). (C) Relative protein levels by western blot in J774A.1 macrophages transfected with mimics of miR-142-3p or not, and that of internalized mycobacteria (MOI 10) after a 1-h challenge. N-Wasp levels are relative to that of α/β-Tubulin. A representative blot from three independent experiments is shown with the densitometry quantification: quantification of the relative levels of N-Wasp in infected macrophages, treated with either mimics of miR-142-3p or scramble (*P ≤ 0.01; **P ≤ 0.001).
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Figure 2: N-Wasp is a target of miR-142-3p. (A) Luciferase assay showing the specific targeting of the 3′UTR of the mRNA of Wasl by the miR-142-3p. Data are represented as the mean fold change per sample ± SD (*P ≤ 0.01). (B) Relative expression of miR-142-3p in J774A.1 macrophages infected with M. smegmatis or M. tuberculosis (MOI 10), as measured by the EXIQON (DK) microRNA qPCR services. Data is represented as the mean fold change per sample ± SD at 1, 4, and 24 h post-infection (*P ≤ 0.05 relative to control). (C) Relative protein levels by western blot in J774A.1 macrophages transfected with mimics of miR-142-3p or not, and that of internalized mycobacteria (MOI 10) after a 1-h challenge. N-Wasp levels are relative to that of α/β-Tubulin. A representative blot from three independent experiments is shown with the densitometry quantification: quantification of the relative levels of N-Wasp in infected macrophages, treated with either mimics of miR-142-3p or scramble (*P ≤ 0.01; **P ≤ 0.001).

Mentions: Given its involvement during early events of phagocytosis (McGee et al., 2001; Caron et al., 2006; Park and Cox, 2009; Dart et al., 2012), and the strong prediction for it being a gene target for miR-142-3p (Tables 1 and 3), we asked whether this miRNA may target the 3′-UTR mRNAs Wasl (mRNA for N-Wasp). To accomplish this, we used a dual luciferase reporter vector system in which the 3′-UTR sequence for Wasl was inserted into the pmirGLO dual-luciferase target expression vector. The system allows one to quantitatively assessing the transcript activity, upon binding to a potential miRNA target, thus validating the specificity of miRNA-mRNA 3′-UTR pair interaction. As shown in Figure 2A, the relative luciferase activity is lower in the pair miR-142-3p/Wasl relative to the control, indicating that the miR-142-3p targets the Wasl mRNA 3′-UTR with high probability (P < 0.01). By contrast, when a plasmid bearing a mutated form of the Wasl sequence by one nucleotide substitution was tested, there was no decrease in luciferase activity detected. Altogether, this demonstrates that Wasl is indeed a target of miR-142-3p.


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.

Bettencourt P, Marion S, Pires D, Santos LF, Lastrucci C, Carmo N, Blake J, Benes V, Griffiths G, Neyrolles O, Lugo-Villarino G, Anes E - Front Cell Infect Microbiol (2013)

N-Wasp is a target of miR-142-3p. (A) Luciferase assay showing the specific targeting of the 3′UTR of the mRNA of Wasl by the miR-142-3p. Data are represented as the mean fold change per sample ± SD (*P ≤ 0.01). (B) Relative expression of miR-142-3p in J774A.1 macrophages infected with M. smegmatis or M. tuberculosis (MOI 10), as measured by the EXIQON (DK) microRNA qPCR services. Data is represented as the mean fold change per sample ± SD at 1, 4, and 24 h post-infection (*P ≤ 0.05 relative to control). (C) Relative protein levels by western blot in J774A.1 macrophages transfected with mimics of miR-142-3p or not, and that of internalized mycobacteria (MOI 10) after a 1-h challenge. N-Wasp levels are relative to that of α/β-Tubulin. A representative blot from three independent experiments is shown with the densitometry quantification: quantification of the relative levels of N-Wasp in infected macrophages, treated with either mimics of miR-142-3p or scramble (*P ≤ 0.01; **P ≤ 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: N-Wasp is a target of miR-142-3p. (A) Luciferase assay showing the specific targeting of the 3′UTR of the mRNA of Wasl by the miR-142-3p. Data are represented as the mean fold change per sample ± SD (*P ≤ 0.01). (B) Relative expression of miR-142-3p in J774A.1 macrophages infected with M. smegmatis or M. tuberculosis (MOI 10), as measured by the EXIQON (DK) microRNA qPCR services. Data is represented as the mean fold change per sample ± SD at 1, 4, and 24 h post-infection (*P ≤ 0.05 relative to control). (C) Relative protein levels by western blot in J774A.1 macrophages transfected with mimics of miR-142-3p or not, and that of internalized mycobacteria (MOI 10) after a 1-h challenge. N-Wasp levels are relative to that of α/β-Tubulin. A representative blot from three independent experiments is shown with the densitometry quantification: quantification of the relative levels of N-Wasp in infected macrophages, treated with either mimics of miR-142-3p or scramble (*P ≤ 0.01; **P ≤ 0.001).
Mentions: Given its involvement during early events of phagocytosis (McGee et al., 2001; Caron et al., 2006; Park and Cox, 2009; Dart et al., 2012), and the strong prediction for it being a gene target for miR-142-3p (Tables 1 and 3), we asked whether this miRNA may target the 3′-UTR mRNAs Wasl (mRNA for N-Wasp). To accomplish this, we used a dual luciferase reporter vector system in which the 3′-UTR sequence for Wasl was inserted into the pmirGLO dual-luciferase target expression vector. The system allows one to quantitatively assessing the transcript activity, upon binding to a potential miRNA target, thus validating the specificity of miRNA-mRNA 3′-UTR pair interaction. As shown in Figure 2A, the relative luciferase activity is lower in the pair miR-142-3p/Wasl relative to the control, indicating that the miR-142-3p targets the Wasl mRNA 3′-UTR with high probability (P < 0.01). By contrast, when a plasmid bearing a mutated form of the Wasl sequence by one nucleotide substitution was tested, there was no decrease in luciferase activity detected. Altogether, this demonstrates that Wasl is indeed a target of miR-142-3p.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Centro de Patogénese Molecular, Faculdade de Farmácia, Unidade dos Retrovírus e Infecções Associadas e Instituto de Medicina Molecular, Universidade de Lisboa Lisboa, Portugal.

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.

Show MeSH
Related in: MedlinePlus