Characterization of MDAΦ, a temperate filamentous bacteriophage of Neisseria meningitidis

Abstract

The mechanism by which Neisseria meningitidis becomes invasive is not well understood. Comparative genomics identified the presence of an 8 kb island in strains belonging to invasive clonal complexes. This island was designated MDA for meningococcal disease associated. MDA is highly conserved among meningococcal isolates and its analysis revealed a genomic organization similar to that of a filamentous prophage such as CTXW of Vibrio cholerae. Subsequent molecular investigations showed that the MDA island has indeed the characteristics of a filamentous prophage, which can enter into a productive cycle and is secreted using the type IV pilus (tfp) secretin PilQ. At least three genes of the prophage are necessary for the formation of the replicative cytoplasmic form (orf1, orf2 and orf9). Immunolabelling of the phage with antibodies against the major capsid protein, ORF4, confirmed that filamentous particles, about 1200 nm long, covered with ORF4 are present at the bacterial surface forming bundles in some places and interacting with pili. The MDA bacteriophage is able to infect different N. meningitidis strains, using the type IV pili as a receptor via an interaction with the adsorption protein ORF6. Altogether, these data demonstrate that the MDA island encodes a functional prophage able to produce infectious filamentous phage particles.

A virulence-associated filamentous bacteriophage of Neisseria meningitidis increases host-cell colonisation

“…Experiments were performed using strains deleted in two genes that have been shown to be important for phage replication and the production of viruses, MDAorf1 and MDAorf9 (S1 Fig) [5, 7]. Strains Z5463 gfp Δ orf1 and Z5463 gfp Δ orf9 described in the material and methods section are unable to produce replicative cytoplasmic forms of the phage, and infectious particle, even though they carry a prophage in their genome.…”

“…The pili encoded by this variant can be labelled by a monoclonal antibody, 20D9. The construction of this strain designated Z5463(SB- aph3’ ) has been previously reported [7]. Bacteria colonizing a FaDu epithelial cell monolayer in a laminar flow chamber were harvested by aspiration as described in the material and methods section.…”

“…This step removed most of the bacteria colonizing the epithelial cells, leaving only bacteria strongly adhering to the epithelial monolayer. Both adhering bacteria and bacteria peeled off the epithelial cells were labelled using the 20D9 monoclonal antibody and a polyclonal antibody directed against the MDAORF4, the major capsid protein [7]. As shown Fig 5A, bacteria obtained from the aspiration were heavily labelled by the polyclonal antibody against the major phage capsid protein, but almost no labelling was obtained using the 20D9 anti-pili monoclonal antibody.…”

“…Z5463(SB- aph3’ ) was obtained following transformation of DNA of strain Nm 8013 SB- aph3’ [35] into Z5463 and selecting for Km resistance [7]. …”

“…This study brought to light a sequence of 8 kb, designated MDA for Meningococcal Disease Associated island, which is associated with an increase ability of invasive disease [5, 6]. Subsequent studies have demonstrated that the MDA island encodes a functional filamentous prophage, designated MDAΦ, able to produce infectious filamentous phage particles [7] (S1 Fig highlights the organization of the MDAΦ genome). However the mechanism by which the MDAΦ prophage increases bacterial invasiveness remains unknown.…”