Role of Rv3351c in trafficking Mycobacterium tuberculosis bacilli in alveolar epithelial cells and its contribution to disease

Although interactions with alveolar macrophages have been well characterized for Mycobacterium tuberculosis, the roles epithelial cells play during infection and disease development have been less studied. We have previously shown that deletion of gene rv3351c reduces M. tuberculosis replication in and necrosis of A549 human type II pneumocyte cells. In the present study, we report that rv3351c is required for lipid raft aggregation on A549 cell plasma membranes during M. tuberculosis infection. Lipid raft aggregation was also induced directly by recombinant Rv3351c protein. A Δrv3351c deletion mutant was less effective than wild type M. tuberculosis at circumventing phagolysosome fusion in A549 cells as evidenced by increased co-localization with lysosomal markers LAMP-2 and cathepsin-L by the mutant bacilli. These observations indicate a role for Rv3351c in modification of the plasma membrane to facilitate trafficking and survival of M. tuberculosis bacilli through alveolar epithelial cells, and support the hypothesis that M. tuberculosis has mechanisms to target the alveolar epithelium. Preliminary data also demonstrate that like the type II pneumocyte-targeting M. tuberculosis secreted protein heparin-binding filamentous hemagglutinin (HBHA), Rv3351c is detected by the host cellular and humoral immune responses during infection, and may play an important role in mycobacterial dissemination from the lungs. Author summary Mycobacterium tuberculosis is the leading causes of death due to a single infectious agent and many facets regarding the pathogenesis of this organism remain unknown. This facultative intracellular bacterial pathogen often establishes infection through inhalation of the bacilli into the alveoli of the lungs. Interactions with alveolar macrophages have been well characterized and it had been assumed that these interactions with phagocytic cells primarily determine the fate of the disease. However, alveolar epithelial cells, such as type II pneumocytes, play important roles in disease progression of other bacterial and viral respiratory pathogens, which provided the impetus to more-closely examine pneumocyte-M. tuberculosis interactions. We describe in this study the role of the M. tuberculosis rv3351c gene product in the internalization and survival of this pathogen in human type II pneumocytes. We previously showed that a Δrv3351c mutant replicates less efficiently and generates less necrosis than the parental M. tuberculosis strain in this cell type. We demonstrate herein that Rv3351c protein induces lipid raft aggregation on the membranes of alveolar epithelial cells and that M. tuberculosis Δrv3351c traffics through LAMP-2-labeled endosomes 30% more frequently than the parent strain. This trafficking toward phagolysosomes may underlie the reduced replication and cytotoxicity of the mutant. The role of Rv3351c in trafficking and survival of M. tuberculosis bacilli through epithelial cells ultimately resulting in dissemination from the lungs may begin with modifications to the plasma membrane prior to attachment. Such a mechanism of activity suggests Rv3351c as a potential vaccine target to train the host immune system to bind and eliminate the protein before it modulates the alveolar epithelium.


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Mycobacterium tuberculosis, the causative agent of tuberculosis infects an estimated one-quarter 73 of the world's population with 60-90% of these individuals potentially harboring latent infection

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(1,2). To date, no vaccine reproducibly protects against the pulmonary form of the disease in post-75 adolescents.

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The alveolar macrophage is generally believed to control the initial success or failure of

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The present study examines attachment, internalization and trafficking within type II

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To examine conversion of LC3 forms, A549 cells were incubated for 12 or 24 hours with 226 M. tuberculosis Erdman or Δrv3351c at a MOI of 100. After extracellular bacteria were removed, 227 Autophagy levels were analyzed by LC3 immunoblotting assay with GAPDH as a loading control 228 ( Figure 7A and B). The LC3-II/GAPDH ratio is higher after Δrv3351c or Erdman infection than 229 uninfected controls at 12 hpi ( Figure 7A and C); however, the LC3-II/GAPDH ratio is higher at 12 230 hpi for A549 cells infected with Δrv3351c than Erdman or cells induced for autophagy by amino    We previously demonstrated microscopically that viable M. tuberculosis Erdman bacilli induce 302 lipid raft aggregation on infected A549 cells to similar or greater levels than lipid raft super 303 aggregator LLO (9). Additionally, we showed that culture filtrates from infected A549 cells also 304 induce lipid raft aggregation when added to fresh monolayers indicating the responsible factor is 305 mycobacterial and likely secreted during or prior to infection (9). In this current study, we 306 demonstrate that infection with Δrv3351c cells did not as efficiently aggregate lipid rafts compared 307 to the parent strain, but recombinant Rv3351c protein alone induced similar levels of lipid raft 308 aggregation on A549 cell plasma membranes as the LLO positive control when added at equal 309 concentrations ( Figure 1). Interestingly, the difference in lipid raft aggregation between A549 cells 310 infected with the wild type and Δrv3351c bacilli did not appear at the gross level to affect the 311 method or rate of bacterial attachment and internalization, but did alter the intracellular trafficking 312 pattern between the two strains. Thus, the overall studies described here provide for a more 313 thorough understanding of the role Rv3351c in the process of M. tuberculosis attachment, 314 internalization and trafficking within type II pneumocytes. However, these data also contribute to 315 the body of knowledge indicating a larger role for alveolar pneumocytes; they may not simply 316 provide a barrier to infection, but may also contribute to the pathology associated with tuberculosis 317 (34-37). What was surprising is the apparent role this protein plays in modifying the host cell 318 membrane to induce lipid raft aggregation, and thus plays a major role in the optimal trafficking 319 that is not observed with the mutant strain; potentially causing the mutant bacteria to attach to, 320 enter the pneumocyte, and traffic through a sub-optimal pathway that is sub-optimal for bacterial 321 survival and spread.

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The process of attachment and internalization is readily observed with both M.