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 8

Exacerbation of mycobacterial infection by pretreatment with isolated vesicles.

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Exacerbation of mycobacterial infection by pretreatment with isolated ve...
(A) Representative images of H&E-stained lung sections of mice that received i.t. infusion of purified BCG MVs or PBS, followed by aerosol challenge with Mtb H37Rv 15 days later. Lungs were analyzed at 15 and 30 days after challenge. (B) Representative images of H&E-stained lung sections of BCG-vaccinated WT C57BL/6 mice infused with vesicles or PBS and challenged with a low dose of H37Rv (50–100 CFUs). Mice were vaccinated with BCG by intradermal injection and 6 weeks later received i.t. PBS (control) or purified BCG MVs. Aerosol challenge with MTb H37Rv was done 15 days later. Lungs were analyzed at 30 days after the challenge. All pictures were taken using a digital camera Coolscope (Nikon). Original magnification, ×40. (C) Quantification of the number of granuloma per lung in mice receiving the indicated treatments followed by Mtb H37Rv challenge. Analysis was done at day 30 after challenge. (D and E) CFUs in lungs (D) and spleens (E) of mice receiving the indicated treatments followed by Mtb H37Rv challenge. Analysis was done at day 30 after challenge. Experimental data are representative of 2 independent experiments. Data represent mean ± SEM. **P < 0.01.