Genomic and biochemical description of class II pilin expressing strains isolated at different sites and at different times.

<p>(A) Genetic organization of the pilin locus in the 8013, FAM20, LIM707, and LIM534 strains. (B) Genetic organization of the core <i>pgl</i> locus in the same strains. (C) High resolution intact protein mass profiling of 4 class II pilin expressing strains demonstrating multisite glycosylation. (D) Alignment of the pilin sequences from collected class II pilin expressing clinical strains. Sequences of the <i>N</i>. <i>meningitidis</i> 8013 and FAM20 strains and the <i>N</i>. <i>gonorrhoeae</i> MS11 strain [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005162#ppat.1005162.ref035" target="_blank">35</a>] are included for reference. Glycosylation sites irrespective of their nature appear as black hexagons.</p

Analysis of the posttranslational modifications of the class II pilin expressing strain FAM20.

<p>(A) Whole protein analysis of type IV pili prepared from the FAM20 strain and mutants of the same strain <i>pptA</i>, <i>pglH</i> and the double mutant <i>pglHpptA</i>. (B) Top-down fragmentation maps of the three main proteoforms found in the FAM20<i>pglHpptA</i> strain showing identified PTMs localized to sites on the PilE primary structure.</p

Oligonucleotides used in this study.

<p>* Restriction sites in bold</p><p>Oligonucleotides used in this study.</p

Interaction diagrams of compounds 6-<i>α</i>-aminocholestanol (20), 6-<i>β</i>-aminocholestanol and 6-ketocholestanol (48) (from left to right) with pocket P2 on apo-LieIF, in the non-phosphorylated form.

<p>Hydrogen bonds are shown in green dashed lines along with their length in Å. Residues establishing hydrophobic interactions with the ligands atoms are shown in red incomplete circles. Ligand atoms of the compound that are implicated in these hydrophobic interactions are also surrounded by red sticks.</p

Complete description of the 23 proteoforms expressed by the FAM20 strain.

<p>Complete description of the 23 proteoforms expressed by the FAM20 strain.</p

LieIF models and pockets.

<p>(a) Apo-LieIF corresponds to the open conformation of LieIF and (b) Holo-LieIF corresponds to its closed conformation. The blue to red color gradient goes from the N-terminus to the C-terminus. The dumbbell shape, consisting of the two linked RecA-like domains that are common to the DBPs, is obtained for both models. (c) Apo-LieIF_trunc/MD model with a representation of the conserved motifs and the identified pockets. Pocket P1 is the orange grid. Pocket P2 is the blue grid. Conserved motifs of the DEAD-box family are shown in different colors: Q-motif in red, motif I in yellow, motif Ia in green, GG doublet in yellow, motif Ib in blue, motif II in magenta, motif III in orange, motif IV in red, QxxR motif in blue, motif V in green and motif VI in yellow. The phosphorylation site (T135) observed in an amastigote version of LieIF is shown in magenta.</p

Summary of the experimental results of the ATPase assays and the <i>in vitro</i> effects on the viability of the promastigotes, THP-1 cells and intramacrophage amastigotes of the three hits.

<p>SI is the ratio between the CC₅₀ value against THP-1 cells and the IC₅₀ value against <i>Leishmania</i> parasites (promastigotes or amastigotes, accordingly).</p

Summary of the number of glycosylation sites found in the different <i>Neisseria meningitidis</i> strains.

<p>Summary of the number of glycosylation sites found in the different <i>Neisseria meningitidis</i> strains.</p

Measured neutral monoisotopic masses of PilE proteoforms from the FAM20<i>pglHpptA</i> double mutant and comparison with theoretical masses calculated from the FAM20 sequence plus indicated PTMs.

<p>* C-C, disulfide bond; PG, phosphoglycerol; GATDH, glyceramido trideoxyhexose</p><p>Measured neutral monoisotopic masses of PilE proteoforms from the FAM20<i>pglHpptA</i> double mutant and comparison with theoretical masses calculated from the FAM20 sequence plus indicated PTMs.</p

Genetically modified strains used in this study.

<p>Genetically modified strains used in this study.</p