Impact of protein domains on PE_PGRS30 polar localization in Mycobacteria.
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.
Institute of Microbiology, Universita' Cattolica del Sacro Cuore, Rome, Italy.
Flavio
F
De Maio
Institute of Physics, Universita' Cattolica del Sacro Cuore, Rome, Italy.
Giuseppe
G
Maulucci
Institute of Microbiology, Universita' Cattolica del Sacro Cuore, Rome, Italy.
Mariachiara
M
Minerva
Department of Molecular Medicine, University of Padua, Padua, Italy.
Saber
S
Anoosheh
Institute of Microbiology, Universita' Cattolica del Sacro Cuore, Rome, Italy.
Ivana
I
Palucci
Institute of Microbiology, Universita' Cattolica del Sacro Cuore, Rome, Italy.
Raffaella
R
Iantomasi
Institute of Physics, Universita' Cattolica del Sacro Cuore, Rome, Italy.
Valentina
V
Palmieri
Institute of Microbiology, Universita' Cattolica del Sacro Cuore, Rome, Italy.
Serena
S
Camassa
Institute of Microbiology, Universita' Cattolica del Sacro Cuore, Rome, Italy.
Michela
M
Sali
Institute of Microbiology, Universita' Cattolica del Sacro Cuore, Rome, Italy.
Maurizio
M
Sanguinetti
Department of Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam, Netherlands.
Wilbert
W
Bitter
Department of Molecular Medicine, University of Padua, Padua, Italy.
Riccardo
R
Manganelli
Institute of Physics, Universita' Cattolica del Sacro Cuore, Rome, Italy.
Marco
M
De Spirito
Institute of Microbiology, Universita' Cattolica del Sacro Cuore, Rome, Italy.
Giovanni
G
Delogu
D020816:Amino Acid Motifs; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D008264:Macrophages / Q000382:microbiology; D051379:Mice; D008969:Molecular Sequence Data; D009163:Mycobacterium bovis / Q000235:genetics / Q000378:metabolism; D020102:Mycobacterium smegmatis / Q000235:genetics / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014774:Virulence
D001426:Bacterial Proteins; D011994:Recombinant Proteins