PPE64 is a mycomembrane channel protein that functions in heme iron uptake and moonlights in biofilm formation in <i>Mycobacterium tuberculosis</i>

Mycobacterium tuberculosis ( Mtb ) is the leading cause of human deaths by an infectious agent. To survive in the human host, Mtb must acquire essential iron nutrients from the host and evade the immune response. In diderm bacteria like Mtb , outer membrane channel transporter proteins are fundamental for nutrient acquisition and immune evasion. Recently, we demonstrated that the Mtb outer mycomembrane PPE64 is a channel protein, providing the first direct evidence of channel-forming capability by a protein of the PPE (proline-proline-glutamate motif) family, which are found exclusively in mycobacteria. Here, we demonstrate that the PPE64 channel protein is specifically required for the uptake of heme, which is the largest source of iron in the human host. Furthermore, PPE64 plays a crucial role in biofilm formation, and this function is not dependent on the iron source in the medium. The moonlighting function of PPE64 is reflected in its two distinct oligomeric states, where the higher-order oligomer specifically forms water-filled channels in membranes and binds heme. The discrete roles of PPE64 are also important in an ex vivo setting, as its absence significantly impairs Mtb growth within human monocytes and alveolar macrophages. Thus, our study reveals that the PPE64 channel protein plays multiple roles in Mtb physiology and establishes it as an important factor in Mtb virulence.IMPORTANCEThe success of any invading bacteria to survive within the host is dictated by their ability to acquire nutrients and overcome the host immune response. Bacterial cell surface proteins play critical roles in these processes at the host-pathogen interface. Here, we show that the PPE64 mycomembrane channel protein is required for heme iron acquisition and biofilm formation, which are fundamental processes that are of great significance to Mycobacterium tuberculosis ( Mtb ) survival within the host. These discrete functions of PPE64 are dictated by the culturing environment and are important for Mtb growth within human macrophages. These observations support an emerging theme in the Mtb field that the PPE protein family functions in trafficking molecules across the outer mycomembrane and has far-reaching implications for understanding of Mtb physiology.