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.