Indeed, PE_PGRS33 shows immunomodulatory properties thanks to its ability to interact with TLR2, which may trigger macrophage cell death. Since their identification in the Mtb genome, PE_PGRS proteins have been implicated in the mechanism of pathogenesis of TB and included in an hypothetical panel of surface mycobacterial antigens involved in immune evasion strategies. In this study, using a panel of GFP-tagged proteins, we investigate the localization of PE_PGRS30 in three mycobacterial species and analyzed the impact of the different protein domains on protein polarization on the bacterial cells. We show that both the PGRS and C-terminal unique domain of PE_PGRS30 contribute to protein localization and that the C-terminal domain is not available on the mycobacterial surface. Moreover, using GFP-protein chimeras we demonstrate that PE_PGRS30 localize at the bacterial poles during infection in macrophages, while PE_PGRS33 remains homogeneously distributed on the mycobacterial surface. These results provide further insights on PE_PGRS protein localization and suggest the functional diversity between PE_PGRS proteins. It remains to be seen whether polarization of PE_PGRS30 is dependent upon interaction with ESX-5 components and expression of the chimeras used in this study in Mtb DESX5 mutants will shed light on the molecular mechanism of this process. The fact that a similar pattern of protein localization for PE_PGRS30 and its functional deletion chimeras was observed in Mtb and BCG suggests that lack of ESX-1 or other region of deletions in BCG does not impact PE_PGRS30 localization. PE_PGRS30 polarization was observed also for the 30PE_PGRSDCTGFP, indicating that the unique 306 amino acids C-terminal domain is not necessary for proper localization of the protein on the mycobacterial cell wall. These results are in line with our previous finding that the C-terminal unique domain is dispensable for the PE_PGRS30-dependent virulence and imply that the PGRS domain is properly exposed or available to deploy its LDN-193189 ALK inhibitor function in Mtb regardless of the C-terminal domain. Concentration at one bacterial pole of proteins and enzymes involved in peptidoglycan synthesis and virulence, such as the ESX1 secretory apparatus is known to be important in mycobacteria. Concentration at a pole of virulence associated proteins or protein scaffolds may be a key step to evade from phagosome or eject from host cells. Indeed, the ESX1 T7SS apparatus was shown to accumulate at the bacterial pole and this process may be instrumental to produce holes in the phagosome that warrant cytoplasm access to Mtb. Our finding indicating that PE_PGRS30 strongly accumulate at the bacterial poles in Mtb infecting macrophages and replicating intracellularly suggests that polarization may be a key step in the PE_PGRS30-dependent virulence mechanism. Since PE_PGRS30 is required for the survival and replication of Mtb in macrophages, it may be hypothesized that PE_PGRS30, alone or in combination with other yet undefined effectors, concentrates at one bacterial pole to maximize its activity. Conversely, PE_PGRS33GFP was homogeneously distributed throughout the bacterial cells during Mtb infection in macrophages.