title: The naturally competent strain Streptococcus thermophilus LMD-9 as a new tool to anchor heterologous proteins on the cell surface.
abstract: BACKGROUND
  From fundamental studies to industrial processes, synthesis of heterologous protein by micro-organisms is widely employed. The secretion of soluble heterologous proteins in the extracellular medium facilitates their recovery, while their attachment to the cell surface permits the use of the recombinant host cells as protein or peptide supports. One of the key points to carry out heterologous expression is to choose the appropriate host. We propose to enlarge the panel of heterologous secretion hosts by using Streptococcus thermophilus LMD-9. This lactic acid bacterium has a generally recognised as safe status, is widely used in the manufacture of yogurts, fermented milks and cheeses, and is easy to transform by natural competence. This study demonstrates the feasibility of secretion of a heterologous protein anchored to the cell surface by S. thermophilus. For this, we used the cell envelope proteinase (CEP) PrtH of Lactobacillus helveticus CNRZ32 CIRM-BIA 103.
  
  
  RESULTS
  Using S. thermophilus LMD-9 as the background host, three recombinant strains were constructed: i) a negative control corresponding to S. thermophilus PrtS- mutant where the prtS gene encoding its CEP was partially deleted; ii) a PrtH+ mutant expressing the L. helveticus PrtH pro-protein with its own motif (S-layer type) of cell-wall attachment and iii) a PrtH+WANS mutant expressing PrtH pro-protein with the LPXTG anchoring motif from PrtS. The PrtH+ and PrtH+WANS genes expression levels were measured by RT-qPCR in the corresponding mutants and compared to that of prtS gene in the strain LMD-9. The expression levels of both fused prtH CEPs genes, regardless of the anchoring motif, reached up-to more than 76% of the wild-type prtS expression level. CEPs were sought and identified on the cell surface of LMD-9 wild-type strain, PrtH+ and PrtH+WANS mutants using shaving technique followed by peptide identification with tandem mass spectrometry, demonstrating that the heterologous secretion and anchoring of a protein of more than 200 kDa was efficient. The anchoring to the cell-wall seems to be more efficient when the LPXTG motif of PrtS was used instead of the S-layer motif of PrtH.
  
  
  CONCLUSIONS
  We demonstrated S. thermophilus LMD-9 could heterologously secrete a high molecular weight protein and probably covalently anchor it to the cell-wall.
authors.forename: Xavier
authors.initials: X
authors.lastname: Lecomte
authors.forename: Valérie
authors.initials: V
authors.lastname: Gagnaire
authors.forename: Valérie
authors.initials: V
authors.lastname: Briard-Bion
authors.forename: Julien
authors.initials: J
authors.lastname: Jardin
authors.forename: Sylvie
authors.initials: S
authors.lastname: Lortal
authors.forename: Annie
authors.initials: A
authors.lastname: Dary
authors.affiliation: Unité de Recherche Animal et Fonctionnalités des Produits Animaux, Equipe Protéolyse et Biofonctionnalité des Protéines et des Peptides, Université de Lorraine, Vandœuvre-lès-Nancy F-54506, France. magali.genay@univ-lorraine.fr.
authors.forename: Magali
authors.initials: M
authors.lastname: Genay
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002473:Cell Wall / Q000378:metabolism; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D052198:Lactobacillus helveticus / Q000201:enzymology; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D048249:Streptococcus thermophilus / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D010450:Endopeptidases; C120659:PrtH protein, Lactobacillus; C417204:PrtS protein, Streptococcus thermophilus; D012697:Serine Endopeptidases