Sequence analysis of pilin conversion events.

<p>(A) Neighbor joining tree of Pilin amino acid sequences of individual colonies picked from WUE4558 and clones 2 and 77. The tree was computed with MEGA5 (<a href="http://www.megasoftware.net/" target="_blank">http://www.megasoftware.net/</a>; p-distance method; 168 positions of the aligned dataset). A bootstrap test was applied (2000 replicates). (B) Deduced amino acid sequences of the hypervariable region D flanked by two cysteine residues <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045132#pone.0045132-Marceau1" target="_blank">[32]</a>. A lysine residue previously associated to pilus bundle formation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045132#pone.0045132-Marceau1" target="_blank">[32]</a> is highlighted by boxes.</p

Presence of LPS immunotypes L1, L3 and L8 in ST-41/44 cc and ST-32 cc meningococci.

<p>Meningococcal LPS was analyzed by immuno dot blots. The isolates were obtained from invasive disease (German reference laboratory for meningococci) and healthy carriers <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045132#pone.0045132-Claus1" target="_blank">[28]</a>, respectively.</p

Mutant class I: increased expression of Opc by clones with enhanced serum resistance.

<p>(<b>A</b>) Coomassie blue stained SDS PAGE showing a prominent band at 26–28 kDa (arrow) in one of the resistant clones (clone 1). WUE4558 is the parental strain. (<b>B</b>) Detection of enhanced Opc-expression in clone 1 by Western blot developed with the Opc-specific monoclonal antibody B306. (<b>C</b>) The number of cytosine residues in the homopolymeric tract of the <i>opc</i> promoter is provided, which has been shown to dictate Opc expression <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045132#pone.0045132-Sarkari1" target="_blank">[24]</a>.</p

Mutant class II: demonstration of LPS immunotype changes and their consequences with regards to serum resistance.

<p>(A) Tricine gel electrophoresis revealed a double banding pattern for clone 53 and clone 62. As controls, strain MC58 (immunotype L3) and its <i>pgm</i> and <i>lgtA</i> (immunotype L8) mutants are shown, which have a truncation of four and two sugar residues, respectively. Clone 77 is shown as a further control, whose increased serum resistance is associated to pilin conversion, but not immunotype switches. (B) Flow cytometry analysis showing reduced deposition of membrane attack complex upon serum stress in strains DE9686 <i>siaD</i>- <i>fhbp</i>- <i>lgtA</i>- and DE9686 <i>siaD</i>- <i>fhbp</i>- l<i>gtA</i>- pAP1<i>lgtC_#53</i> in comparison to strain DE9686 <i>siaD</i>-<i>fhbp</i>-. (C) Resistance towards 10% NHS was significantly enhanced in DE9686 <i>siaD</i>- <i>fhbp</i>- l<i>gtA</i>- and DE9686 <i>siaD</i>- <i>fhbp</i>- <i>lgtA</i>- pAP1<i>lgtC_#53</i>, when compared to DE9686 <i>siaD</i>- <i>fhbp</i>-. Data were compared by paired student's t-test. (D) Immuno dot blots demonstrating the immunotypes of clones 53 and 62 both expressing L1 and L8 LPS. The structure of the terminal sugar residues of meningococcal LPS is schematically depicted for the three type strains (WUE4558, L3; WUE679, L1 type strain; WUE2535, lgtA knock out strain, L8; for further information see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045132#pone-0045132-t001" target="_blank">Table 1</a>).</p

Mutant class III: antigenic variation of pilin and its impact on autoaggregation.

<p>(<b>A</b>) Immunoblot demonstrating class II pilin by antibody SM1(arrows). Both clones 2 and 77 revealed altered migration of PilE. (B) Autoaggregation assay: both clones 2 and 77 show accelerated rates of autoaggregation. Bacterial suspensions were kept without agitation for 150 min and the optical density was measured repeatedly to assess clearance of the suspension by autoaggregation and sedimentation.</p

Analysis of complement attack by flow cytometry of isogenic derivatives of DE9686 <i>siaD</i>- <i>fhbp</i>-.

<p>(<b>A</b>) Enhanced vitronectin binding in the complemented mutant overexpressing Opc (DE9686 <i>siaD</i>- <i>fhbp</i>- <i>opc</i>- pAP1<i>opc</i>). (<b>B</b>) Membrane attack complex deposition on the Opc-positive strains DE9686 <i>siaD</i>- <i>fhbp</i>- and DE9686 <i>siaD</i>- <i>fhbp</i>- <i>opc</i>- pAP1<i>opc</i> in comparison to that on the <i>opc</i> knock out strain. (<b>C</b>) C3d load in DE9686 <i>siaD-, fhbp</i>- and the corresponding <i>opc</i> knock and <i>opc</i> complemented strain (<b>D</b>) Resistance towards 10% NHS of DE9686 <i>siaD</i>- <i>fhbp</i>-, DE9686 <i>siaD</i>- <i>fhbp</i>- <i>opc</i>-, and DE9686 <i>siaD</i>- <i>fhbp</i>- <i>opc</i>- pAP1<i>opc</i>. Data were compared by paired student's t-test.</p

Serum killing assay of representative clones belonging to mutant classes I through III.

<p>The data display means of two to three independent experiments. (A) Clones 1 and 14 represent mutant class I overexpressing the Opc protein (n = 31); (B) Clones 53 and 62 (mutant class II, n = 2) displayed altered LPS phenotype compared to the parental strain WUE4558; (C) Clones 2 and 77 showed pilin variation (mutant class III, n = 2).</p