Role of Rv3351c in traffickingMycobacterium tuberculosisbacilli in alveolar epithelial cells and its contribution to disease
Authors: Prescott Megan , Fine-Coulson Kari , Metcalfe Maureen , Gupta Tuhina , Dookwah Michelle , Pavlicek Rebecca , Yahyaoui Azami Hind , Reaves Barbara , Hikal Ahmed , Tiemeyer Michael , Karls Russell , Quinn Frederick 
Published: December 2020

Although interactions with alveolar macrophages have been well characterized forMycobacterium tuberculosis, the roles epithelial cells play during infection and disease development have been less studied. We have previously shown that deletion of generv3351creducesM. tuberculosisreplication in and necrosis of A549 human type II pneumocyte cells. In the present study, we report thatrv3351cis required for lipid raft aggregation on A549 cell plasma membranes duringM. tuberculosisinfection. Lipid raft aggregation was also induced directly by recombinant Rv3351c protein. AΔrv3351cdeletion mutant was less effective than wild typeM. tuberculosisat 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 ofM. tuberculosisbacilli through alveolar epithelial cells, and support the hypothesis thatM. tuberculosishas mechanisms to target the alveolar epithelium. Preliminary data also demonstrate that like the type II pneumocyte-targetingM. tuberculosissecreted 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 tuberculosisis 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. tuberculosisinteractions. We describe in this study the role of theM. tuberculosis rv3351cgene product in the internalization and survival of this pathogen in human type II pneumocytes. We previously showed that aΔrv3351cmutant replicates less efficiently and generates less necrosis than the parentalM. tuberculosisstrain in this cell type. We demonstrate herein that Rv3351c protein induces lipid raft aggregation on the membranes of alveolar epithelial cells and thatM. tuberculosis Δrv3351ctraffics 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 ofM. tuberculosisbacilli 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.