title: Polar localization of PhoN2, a periplasmic virulence-associated factor of Shigella flexneri, is required for proper IcsA exposition at the old bacterial pole.
abstract: Proper protein localization is critical for bacterial virulence. PhoN2 is a virulence-associated ATP-diphosphohydrolase (apyrase) involved in IcsA-mediated actin-based motility of S. flexneri. Herein, by analyzing a ΔphoN2 mutant of the S. flexneri strain M90T and by generating phoN2::HA fusions, we show that PhoN2, is a periplasmic protein that strictly localizes at the bacterial poles, with a strong preference for the old pole, the pole where IcsA is exposed, and that it is required for proper IcsA exposition. PhoN2-HA was found to be polarly localized both when phoN2::HA was ectopically expressed in a Escherichia coli K-12 strain and in a S. flexneri virulence plasmid-cured mutant, indicating a conserved mechanism of PhoN2 polar delivery across species and that neither IcsA nor the expression of other virulence-plasmid encoded genes are involved in this process. To assess whether PhoN2 and IcsA may interact, two-hybrid and cross-linking experiments were performed. While no evidence was found of a PhoN2-IcsA interaction, unexpectedly the outer membrane protein A (OmpA) was shown to bind PhoN2-HA through its periplasmic-exposed C-terminal domain. Therefore, to identify PhoN2 domains involved in its periplasmic polar delivery as well as in the interaction with OmpA, a deletion and a set of specific amino acid substitutions were generated. Analysis of these mutants indicated that neither the (183)PAPAP(187) motif of OmpA, nor the N-terminal polyproline (43)PPPP(46) motif and the Y155 residue of PhoN2 are involved in this interaction while P45, P46 and Y155 residues were found to be critical for the correct folding and stability of the protein. The relative rapid degradation of these amino acid-substituted recombinant proteins was found to be due to unknown S. flexneri-specific protease(s). A model depicting how the PhoN2-OmpA interaction may contribute to proper polar IcsA exposition in S. flexneri is presented.
authors.affiliation: Dipartimento di Scienze Sperimentali e Cliniche, Università "G. D'Annunzio", Chieti, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Scribano
authors.affiliation: Dipartimento di Scienze Sperimentali e Cliniche, Università "G. D'Annunzio", Chieti, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Petrucca
authors.affiliation: Dipartimento di Sanità Pubblica e Malattie Infettive Università "Sapienza" di Roma, Rome, Italy.
authors.forename: Monica
authors.initials: M
authors.lastname: Pompili
authors.affiliation: Dipartimento di Sanità Pubblica e Malattie Infettive Università "Sapienza" di Roma, Rome, Italy.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Ambrosi
authors.affiliation: Dipartimento di Sanità Pubblica e Malattie Infettive Università "Sapienza" di Roma, Rome, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Bruni
authors.affiliation: Dipartimento di Sanità Pubblica e Malattie Infettive Università "Sapienza" di Roma, Rome, Italy.
authors.forename: Carlo
authors.initials: C
authors.lastname: Zagaglia
authors.affiliation: Dipartimento di Biologia e Biotecnologie "C. Darwin", Università Sapienza di Roma, Rome, Italy.
authors.forename: Gianni
authors.initials: G
authors.lastname: Prosseda
authors.affiliation: Dipartimento di Sanità Pubblica e Malattie Infettive Università "Sapienza" di Roma, Rome, Italy.
authors.forename: Lucia
authors.initials: L
authors.lastname: Nencioni
authors.affiliation: Dipartimento di Scienze, Università di "Roma Tre", Rome, Italy.
authors.forename: Mariassunta
authors.initials: M
authors.lastname: Casalino
authors.affiliation: Dipartimento di Scienze, Università di "Roma Tre", Rome, Italy ; Istituto Nazionale di Fisica Nucleare, Sezione di "Roma Tre", Rome, Italy.
authors.forename: Fabio
authors.initials: F
authors.lastname: Polticelli
authors.affiliation: Dipartimento di Scienze Sperimentali e Cliniche, Università "G. D'Annunzio", Chieti, Italy.
authors.forename: Mauro
authors.initials: M
authors.lastname: Nicoletti
mesh_terms: D020816:Amino Acid Motifs; D001081:Apyrase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D019897:Periplasm / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D055550:Protein Stability; D021381:Protein Transport; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012763:Shigella flexneri / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D014157:Transcription Factors / Q000378:metabolism*; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D037521:Virulence Factors; C058483:virG protein, Shigella flexneri; C078167:OMPA outer membrane proteins; D001081:Apyrase