Mycobacteria release active membrane vesicles that modulate immune responses in a TLR2-dependent manner in mice
Rafael Prados-Rosales, Andres Baena, Luis R. Martinez, Jose Luque-Garcia, Rainer Kalscheuer, Usha Veeraraghavan, Carmen Camara, Joshua D. Nosanchuk, Gurdyal S. Besra, Bing Chen, Juan Jimenez, Aharona Glatman-Freedman, William R. Jacobs Jr., Steven A. Porcelli, Arturo Casadevall
Rafael Prados-Rosales, Andres Baena, Luis R. Martinez, Jose Luque-Garcia, Rainer Kalscheuer, Usha Veeraraghavan, Carmen Camara, Joshua D. Nosanchuk, Gurdyal S. Besra, Bing Chen, Juan Jimenez, Aharona Glatman-Freedman, William R. Jacobs Jr., Steven A. Porcelli, Arturo Casadevall
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Research Article Infectious disease

Mycobacteria release active membrane vesicles that modulate immune responses in a TLR2-dependent manner in mice

Abstract

Bacteria naturally release membrane vesicles (MVs) under a variety of growth environments. Their production is associated with virulence due to their capacity to concentrate toxins and immunomodulatory molecules. In this report, we show that the 2 medically important species of mycobacteria, Mycobacterium tuberculosis and Mycobacterium bovis bacille Calmette-Guérin, release MVs when growing in both liquid culture and within murine phagocytic cells in vitro and in vivo. We documented MV production in a variety of virulent and nonvirulent mycobacterial species, indicating that release of MVs is a property conserved among mycobacterial species. Extensive proteomic analysis revealed that only MVs from the virulent strains contained TLR2 lipoprotein agonists. The interaction of MVs with macrophages isolated from mice stimulated the release of cytokines and chemokines in a TLR2-dependent fashion, and infusion of MVs into mouse lungs elicited a florid inflammatory response in WT but not TLR2-deficient mice. When MVs were administered to mice before M. tuberculosis pulmonary infection, an accelerated local inflammatory response with increased bacterial replication was seen in the lungs and spleens. Our results provide strong evidence that actively released mycobacterial vesicles are a delivery mechanism for immunologically active molecules that contribute to mycobacterial virulence. These findings may open up new horizons for understanding the pathogenesis of tuberculosis and developing vaccines.

Authors

Rafael Prados-Rosales, Andres Baena, Luis R. Martinez, Jose Luque-Garcia, Rainer Kalscheuer, Usha Veeraraghavan, Carmen Camara, Joshua D. Nosanchuk, Gurdyal S. Besra, Bing Chen, Juan Jimenez, Aharona Glatman-Freedman, William R. Jacobs Jr., Steven A. Porcelli, Arturo Casadevall

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Figure 1

Dependence of MV production by mycobacteria on cell viability.

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Dependence of MV production by mycobacteria on cell viability. TEM showi...
TEM showing production of MVs by BCG (A and C) or Mtb strain H37Rv (B and D). In A and B, vesicles are seen budding from the surface of the bacteria (arrows). In C and D, arrows indicate vesicles that appear to have separated from the bacterial cell. The inset shows a vesicle associated to the bacterial surface. Scale bars: 50 nm. (E and F) TEM of MVs isolated from a day-14 culture of BCG (E) or Mtb H37Rv (F) grown in MM. Scale bars: 100 nm. The size distribution of isolated vesicles from BCG (G) or Mtb H37Rv (H) was determined by electron microscopy. (I) BCG cultures were labeled with 14C-acetate (2 μCi ml–1), harvested on day 5, and either left untreated or heat inactivated at 80°C for 2 hours. The viability of the cultures was determined by plating on 7H11 solid media at day 10, and percentage of survival was calculated by comparing CFU with bacterial particle counts obtained using a counting chamber. Each point represents an independent culture. (J) Cells prepared as described for I were resuspended in fresh medium and incubated for 5 additional days. The vesicle isolation procedure was carried out on supernatants from 3 independent cultures of heat-killed (80°C 2 hours) or live untreated BCG. The background level of radioactivity was determined isolating MVs on sterile medium containing 14C-acetate (2 μCi ml–1).