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 183PAPAP187 motif of OmpA, nor the N-terminal polyproline 43PPPP46 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.

T Follicular Helper Cells Promote a Beneficial Gut Ecosystem for Host Metabolic Homeostasis by Sensing Microbiota-Derived Extracellular ATP

“…The IgA response to E. coli is dependent on Tfh cells in PPs (Lécuyer et al., 2014) and is significantly more effective in P2rx7 −/− mice (Proietti et al., 2014), suggesting that P2X7 activity can affect the T cell-dependent IgA response. We used a recombinant E. coli K-12 strain carrying the pHND10 plasmid, which encodes phoN2 ::HA fusion of Shigella flexneri , a periplasmic ATP-diphosphohydrolase (apyrase) (Santapaola et al., 2006, Scribano et al., 2014). Extracellular ATP released concomitantly with E. coli growth (Hironaka et al., 2013) was undetectable in cultures of these transformants, indicating that apyrase efficiently abrogated ATP secretion (data not shown).…”

Establishment of a Protein Concentration Gradient in the Outer Membrane Requires Two Diffusion-Limiting Mechanisms

“…The best-studied case is that of the IscA protein of Shigella flexneri, which forms a polar gradient at the old cell pole (54). Polar localization of IcsA is directed by a signal present in the protein, and it depends on OmpA and PhoN (55)(56)(57)(58)(59). It would be interesting to know if OmpA in this bacterium also forms a polar gradient that could direct the insertion of IcsA.…”

The Shigella flexneri OmpA amino acid residues 188 EVQ 190 are essential for the interaction with the virulence factor PhoN2

“…In this respect, we have previously shown that phoN2 , a gene located on a highly conserved region of the virulence plasmid of S. flexneri , encoding a periplasmic ATP-diphosphohydrolase (apyrase), participates, at least indirectly, to proper localization of IcsA at the bacterial surface [1] , [5] . By generating phoN2 ::HA fusions in the wild-type S. flexneri 5a strain M90T, we have shown that periplasmic PhoN2 strictly localizes at the bacterial poles, with a strong preference for the old pole, just beneath IcsA [1] . To evaluate whether PhoN2 and IcsA may interact for the correct exposition of IcsA, we conducted two-hybrid assays in yeast and cross-linking experiments.…”

“…Surprisingly, a strong interaction of PhoN2 with the OM protein A (OmpA) was found. Sequence analysis indicated that OmpA binds PhoN2 through its periplasmic-exposed C-terminal domain (minimal interaction region, amino acid residues 166–250) [1] . OmpA is the major OM protein of Gram-negative bacteria principally involved in the structural integrity of the OM [6] .…”