Phages with tails (Caudovirales) belong to three families, among which Siphoviridae possess a non contractile tail terminated by a tail tip or a baseplate serving for phage adsorption on its host. While the mechanism of bacterial infection by the two other Caudovirales families has been well documented, much less is known concerning infection of gram+ by Siphoviridae.
Our group aims at deciphering the mechanisms underlying infection of Lactococcus lactis by siphophages. L. lactis, a Gram+ bacterium, and its bacteriophages (siphoviridae, dsDNA), are a topic of economical and scientific importance. Lactococcus lactis infection by virulent phages is an economical problem impairing any industrial milk fermentation for cheese production, because virulent phages are ubiquitous within their process environments as well as within pasteurized milk. Besides, several hundreds of L. lactis phages have been isolated worldwide, each phage infecting specifically one or a few L. lactis strains.
In a previous project, we have determined the crystal structures of the receptor binding proteins (RBPs) from lactococcal phages, p2 and TP901-1, because the first step of infection involves the specific interaction of the phage RBP with the saccharidic host receptor evenly distributed at the surface of cell walls. The RBPs are attached to a phage organelle, the baseplate, which orientates it properly for receptor recognition. We have determined the structures of the phages p2 and TP901-1 baseplates by EM and X-ray diffraction. These two organelles (1.1 and 1.9 MDa) harbour 18 and 54 sugar binding sites, respectively. We have shown that p2 baseplate changes conformation in the presence of Ca++, a cation strictly necessary for infection. The 3D knowledge of the components of the baseplates revealed that many of them possess conserved structures. In particular we had shown that a common protein forms a baseplate hub of most gram+ infecting siphophages. Furthermore, some of these structural features are shared with other phages (Myoviridae) or even with the bacterial type VI secretion system.
The present projects aims at keeping the momentum acquired. We have enormously progressed in understanding the mechanism of host recognition, still, we do not understand how the recognition event generates a signal, which, transmitted along the tail, triggers the capsid’s portal protein opening and DNA release. A first possible mechanism involves conformational changes of the major tail proteins (MTPs) induced by the baseplate activation. It might then propagate along the tail up to the terminator, and finally trigger the portal opening. A second hypothesis is based on a bell-ringing mechanism. The central component of the tail, the tail measure protein (TMP), might be pulled by the baseplate movement, which in turn would act on the portal protein. The portal trigger mechanism is likely general among siphophages and extends thus the generality of our project.
Deciphering the trigger mechanism requires approaches in several directions. We will first examine by electron microscopy (EM) the effect of the receptors on the complete phage, ie, the conformational changes of the baseplate and eventually of the tail before and after DNA release. A second approach will involve the production (in E.coli or L.lactis) of complexes larger than the baseplates, incorporating all the components of the baseplate and tail, as well as receptor saccharides. Indeed, the tail size will be decreased to a few hexameric rings of the MTPs by reducing the size of the TMP. These constructions will be examined jointly by cryoEM and X-ray diffraction, in order to determine which of the two above-described mechanism is most likely. The virologists will provide the phages and perform functional infection assays of putatively important mutants identified by structural studies. Our ultimate goal is to assemble sufficient data to present a realistic “film” of viral infection, up to DNA ejection.
Monsieur Christian CAMBILLAU (UNIVERSITE AIX-MARSEILLE II [DE LA MEDITERRANEE]) – firstname.lastname@example.org
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
UNIVMED UNIVERSITE AIX-MARSEILLE II [DE LA MEDITERRANEE]
Université Laval (UL) Université Laval
University College Cork (UCC) University College Cork
INRA Jouy-en-Josas INSTITUT NATIONAL DE LA RECHERCHE AGRONOMIQUE - CENTRE DE RECHERCHE DE JOUY-EN-JOSAS
Help of the ANR 349,991 euros
Beginning and duration of the scientific project: July 2011 - 36 Months