On the killing of mycobacteria by macrophages

Molecular Pathogenesis Centre, Unit of Retrovirus and Associated Infections, Faculty of Pharmacy, University of Lisbon, Av. Forcas Armadas, 1600-083 Lisbon, Portugal.
Cellular Microbiology (Impact Factor: 4.92). 03/2008; 10(2):529-48. DOI: 10.1111/j.1462-5822.2007.01067.x
Source: PubMed


Both pathogenic and non-pathogenic mycobacteria are internalized into macrophage phagosomes. Whereas the non-pathogenic types are invariably killed by all macrophages, the pathogens generally survive and grow. Here, we addressed the survival, production of nitrogen intermediates (RNI) and intracellular trafficking of the non-pathogenic Mycobacterium smegmatis, the pathogen-like, BCG and the pathogenic M. bovis in different mouse, human and bovine macrophages. The bacteriocidal effects of RNI were restricted for all bacterial species to the early stages of infection. EM analysis showed clearly that all the mycobacteria remained within phagosomes even at late times of infection. The fraction of BCG and M. bovis found in mature phagolysosomes rarely exceeded 10% of total, irrespective of whether bacteria were growing, latent or being killed, with little correlation between the extent of phagosome maturation and the degree of killing. Theoretical modelling of our data identified two different potential sets of explanations that are consistent with our results. The model we favour is one in which a small but significant fraction of BCG is killed in an early phagosome, then maturation of a small fraction of phagosomes with both live and killed bacteria, followed by extremely rapid killing and digestion of the bacteria in phago-lysosomes.

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Available from: Luisa Jordao, Oct 07, 2015
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    • "In addition, actin filament assembly also plays a role in the pro-inflammatory response. Several signaling lipids, cAMP, extracellular ATP and the P2X7 receptor, were shown to be involved in actin assembly and the killing/survival of pathogenic mycobacteria (Kalamidas et al., 2006; Treede et al., 2007; Jordao et al., 2008a,b; Kühnel et al., 2008; Kuehnel et al., 2009). Furthermore, some lipid effectors that regulate actin assembly also control NF-κB, a transcription factor involved in the pro-inflammatory response (Gutierrez et al., 2009). "
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    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.
    Frontiers in Cellular and Infection Microbiology 06/2013; 3:19. DOI:10.3389/fcimb.2013.00019 · 3.72 Impact Factor
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    • "resides largely within a phagosome-like compartment of host macrophages during infection (Jordao et al., 2008). Inflammasome activation it is postulated to occur only if these effectors or products from a signalling cascade reach the cytosol and gain access to NLRs. "
    Understanding Tuberculosis - Analyzing the Origin of Mycobacterium Tuberculosis Pathogenicity, 02/2012; , ISBN: 978-953-307-942-4
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    • "We hypothesized that the infection-induced expression of AMPs may be responsible for the observed phenotype. Our quantitative real-time PCR data showed that CRAMP mRNA levels were indeed increased during both killing phases in M. smegmatis-infected J774 macrophages, whereas in RAW macrophages after an initial increase (1–4 h) a decrease in the CRAMP transcripts was observed, which correlated with initial bacterial growth between 1 and 4 h of infection followed by a decline in intracellular survival rate (Jordao et al., 2008). Together, these findings argue that the observed upregulation of cathelicidin during the infection stages most likely contributed to the killing of M. smegmatis. "
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    ABSTRACT: Macrophages have been shown to kill Mycobacterium tuberculosis through the action of the antimicrobial peptide cathelicidin (CAMP), whose expression was shown to be induced by 1,25-dihydroxyvitamin D3 (1,25D3). Here, we investigated in detail the antimycobacterial effect of murine and human cathelicidin against Mycobacterium smegmatis and M. bovis BCG infections. We have synthesized novel LL-37 peptide variants that exhibited potent in vitro bactericidal activity against M. smegmatis, M. bovis BCG and M. tuberculosis H37Rv, as compared with parental peptide. We show that the exogenous addition of LL-37 or endogenous overexpression of cathelicidin in macrophages significantly reduced the intracellular survival of mycobacteria relative to control cells. An upregulation of cathelicidin mRNA expression was observed that correlated with known M. smegmatis killing phases in J774 macrophages. Moreover, RNAi-based Camp knock-down macrophages and Camp(-/-) bone marrow derived mouse macrophages were significantly impaired in their ability to kill mycobacteria. M. smegmatis killing in Camp(-/-) macrophages was less extensive than in Camp(+/+) cells following activation with FSL-1, an inducer of cathelicidin expression. Finally we show that LL-37 and 1,25D3 treatment results in increase in colocalization of BCG-containing phagosomes with lysosomes. Altogether, these data demonstrate that cathelicidin plays an important role in controlling intracellular survival of mycobacteria.
    Cellular Microbiology 07/2011; 13(10):1601-17. DOI:10.1111/j.1462-5822.2011.01644.x · 4.92 Impact Factor
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