Impact of Protein Domains on PE_PGRS30 Polar Localization in Mycobacteria

Abstract

PE_PGRS proteins are unique to the Mycobacterium tuberculosis complex and a number of other pathogenic mycobacteria. PE_PGRS30, which is required for the full virulence of M. tuberculosis (Mtb), has three main domains, i.e. an N-terminal PE domain, repetitive PGRS domain and the unique C-terminal domain. To investigate the role of these domains, we expressed a GFP-tagged PE_PGRS30 protein and a series of its functional deletion mutants in different mycobacterial species (Mtb, Mycobacterium bovis BCG and Mycobacterium smegmatis) and analysed protein localization by confocal microscopy. We show that PE_PGRS30 localizes at the mycobacterial cell poles in Mtb and M. bovis BCG but not in M. smegmatis and that the PGRS domain of the protein strongly contributes to protein cellular localization in Mtb. Immunofluorescence studies further showed that the unique C-terminal domain of PE_PGRS30 is not available on the surface, except when the PGRS domain is missing. Immunoblot demonstrated that the PGRS domain is required to maintain the protein strongly associated with the non-soluble cellular fraction. These results suggest that the repetitive GGA-GGN repeats of the PGRS domain contain specific sequences that contribute to protein cellular localization and that polar localization might be a key step in the PE_PGRS30-dependent virulence mechanism.

The Enigmatic PE/PPE Multigene Family of Mycobacteria and Tuberculosis Vaccination

“…Furthermore, certain PE/PPEs have been shown to aggregate near the poles of mycobacteria similar to ESX1, and this could be related to a specific function in M. tuberculosis (36, 37). Another PE-PGRS, PE-PGRS 30, has also been shown to arrest macrophage acidification and growth of M.…”

PE_PGRS3 ofMycobacterium tuberculosisis specifically expressed at low phosphate concentration, and its arginine-rich C-terminal domain mediates adhesion and persistence in host tissues when expressed inMycobacterium smegmatis

“…To test this hypothesis, recombinant strains were grown until mid‐log phase and then subinoculated in low P i or standard P i Sauton medium and incubated up to 15 days, when bacteria were plated on chamber slides and fixed before confocal fluorescence analysis. The recombinant Ms strains expressing PE_PGRS33 GFP and native GFP under the control of the pe_pgrs33 and hsp60 promoters (Cascioferro et al, ; De Maio et al, ; Delogu et al, ), respectively, were included as controls. Expression of PE_PGRS4 GFP , PE_PGRS33 GFP , and cytosolic GFP was observed in both low and standard P i concentrations, whereas PE_PGRS3 GFP was expressed only when bacteria were cultured in low P i (Figure a).…”

“…Ms mc 2 155 and Mtb H37Rv were grown at 37°C in Middelbrook 7H9 broth medium (difco Becton‐Dickinson), supplemented with 0.2% glycerol (Sigma‐Aldrich), 10% ADC (Becton‐Dichnison), and 0.05% Tween 80 (Sigma‐Aldrich) using standard procedures (De Maio et al, ; Delogu, Pusceddu, et al, ).…”

“…Two hundred‐microlitre concentrated cells were mixed with 1 μg of DNA and incubated for 5 min at room temperature (RT) and then transferred to 0.2‐cm cuvettes (BioRad). Samples were electroporated using an electroporation system Bio‐RAD GenePulser X Cell™ with following parameters: voltage 1,250 V, capacitance 25 μF, and resistance 800 Ω (De Maio et al, ; Palucci et al, ). After the pulse, the cells were recovered in 1 ml of liquid medium, incubated for 4 hr and finally plated on 7H11 as described.…”

Immunoregulatory functions and expression patterns of PE/PPE family members: Roles in pathogenicity and impact on anti‐tuberculosis vaccine and drug design

“…Cell wall localization enables the PE/PPE proteins to elicit potent B‐ and T‐cell responses and also to modulate host immune responses. The PGRS domain of PE_PGRS30, a virulence factor, is responsible for targeting the protein to mycobacterial cell poles in M. tuberculosis and M. bovis . In case of other proteins like PPE17, it is the PPE domain which is crucial for cell surface localization .…”