Tuberculosis (TB) is a highly prevalent human infection and a formidable public health challenge. According to the annual surveys conducted by World Health Organization (WHO), there were an estimated 10 million active TB disease cases which resulted in 1.6 million deaths in 2017, the highest from a single infectious agent [1]. The WHO has also estimated that ~25% of the world population is latently infected with M. tuberculosis and 5%-10% of the infected individuals will eventually develop active TB during their life time. The lifetime risk of active disease in HIV coinfected individuals is ~50% [1]. Also, most of the active disease cases in low TB incidence countries occur in latently infected individuals as a result of reactivation of latent infection [2]. The establishment of infection first requires adherence to and internalization by alveolar macrophages. Although the effector molecules exploited by M. tuberculosis for entry into macrophages are not fully defined, invasin-like membraneassociated Mammalian Cell Entry (Mce) proteins play an important role in facilitating the internalization of mycobacteria into mammalian cells [3]. There are four homologous mce operons (mce1-4) on the M. tuberculosis genome, each encoding six (A-F) Mce proteins. Previous studies carried out on Mce1A have shown that recombinant Escherichia coli cells expressing Mce1A invade and survive inside macrophages and latex beads coated with Mce1A are internalized by non-phagocytic HeLa cells [4]. We have previously shown that latex beads coated with Mce3A and Mce3E are also internalized by mammalian (HeLa) cells [5]. Here we show further evidence that latex beads coated with Mce3A (Figure 1a) and Mce3E (Figure 1b) induce membrane invagination and formation of pedestal-like structures due to elongation of microvilli surrounding the bead. The beads are subsequently phagocytosed by HeLa cells and appear in the cytoplasm either alone or in clusters without being compartmentalized within vacuoles of the HeLa cells.

Figure 1: Transmission electron micrographs showing HeLa cell plasma membrane perturbations and formation of pedestal-like structures due to elongation of microvilli surrounding the latex bead coated with pure Mce3A protein (panel a) and completely internalized latex beads coated with Mce3E by HeLa cells (panel b). The protein-coated latex beads in contact with HeLa cells as well as those internalized by HeLA cells are marked by arrows. The results shown here are representative of two separate experiments.