Expression and assembly of <i>N. meningitidis</i> BamA in <i>E. coli</i>.

<p>A: Expression of Nm<i>bamA</i> in <i>E. coli</i>. <i>E. coli</i> strain UTP_BAD::<i>bamA</i> containing pFP10-Nm<i>bamA</i> was grown in LB containing 0.02% arabinose in the absence or presence of IPTG. Cell envelopes were isolated and analyzed by immunoblotting with Mab α-POTRA1_Nm. B: Assembly of <i>Nm</i>BamA in the <i>E. coli</i> OM. Cell envelopes from <i>E. coli</i> strain UTP_BAD::<i>bamA</i> containing pFP10-Nm<i>bamA</i>grown in LB containing 0.02% arabinose and IPTG were extracted with urea (Ec). As a control, cell envelopes of strain HB-1 were extracted with urea (Nm). Input (i), urea-insoluble (p) and -soluble (s) fractions were analyzed by SDS-PAGE and immunoblot analysis with Mab α-POTRA1_Nm. C: Co-purification of Bam-complex components with His-tagged <i>Nm</i>BamA in <i>E. coli</i>. Cell envelopes of <i>E. coli</i> DH5α cells containing pRV-His-Nm<i>bamA</i> either induced (lanes 2) or not (lanes 1) with IPTG were extracted with Elugent and subjected to Ni²⁺-NTA purification. Shown are elution fractions analyzed by denaturing SDS-PAGE and silver staining (left panel) or immunoblotting (right panels) using antisera against the indicated proteins. As a positive control for BamB detection, cell envelopes derived from strain DH5α were also analyzed on blot (lane c). The arrow indicates the position of BamB.</p

Species-Specificity of the BamA Component of the Bacterial Outer Membrane Protein-Assembly Machinery

<div><p>The BamA protein is the key component of the Bam complex, the assembly machinery for outer membrane proteins (OMP) in gram-negative bacteria. We previously demonstrated that BamA recognizes its OMP substrates in a species-specific manner <i>in vitro</i>. In this work, we further studied species specificity <i>in vivo</i> by testing the functioning of BamA homologs of the proteobacteria <i>Neisseria meningitidis</i>, <i>Neisseria gonorrhoeae</i>, <i>Bordetella pertussis</i>, <i>Burkholderia mallei</i>, and <i>Escherichia coli</i> in <i>E. coli</i> and in <i>N. meningitidis</i>. We found that no BamA functioned in another species than the authentic one, except for <i>N. gonorrhoeae</i> BamA, which fully complemented a <i>N. meningitidis bamA</i> mutant. <i>E. coli</i> BamA was not assembled into the <i>N. meningitidis</i> outer membrane. In contrast, the <i>N. meningitidis</i> BamA protein was assembled into the outer membrane of <i>E. coli</i> to a significant extent and also associated with BamD, an essential accessory lipoprotein of the Bam complex.Various chimeras comprising swapped N-terminal periplasmic and C-terminal membrane-embedded domains of <i>N. meningitidis</i> and <i>E. coli</i> BamA proteins were also not functional in either host, although some of them were inserted in the OM suggesting that the two domains of BamA need to be compatible in order to function. Furthermore, conformational analysis of chimeric proteins provided evidence for a 16-stranded β-barrel conformation of the membrane-embedded domain of BamA. </p> </div

Expression of heterologous <i>bamA</i> variants in <i>N. meningitidis</i> and <i>E. coli</i>.

<p>A, B: <i>N. meningitidis</i> strain HB-1 and its derivatives were grown with IPTG to induce expression of <i>B. pertussis</i> (Bper), <i>B. mallei</i> (Bmal), <i>N. gonorrhoeae</i> (Ngo) and as control, <i>E. coli</i> (Eco) <i>bamA</i> genes from plasmids. A: Cell lysates were analyzed by SDS-PAGE and immunoblotting with the antisera indicated on the right. Note that also chromosome-encoded <i>Nm</i>BamA is detected with the α-N-<i>Nm</i>BamA antiserum in the upper panel. B: RNA was isolated and treated or not with reverse transcriptase (RT). Specific cDNA was detected by conventional PCR followed by agarose gel electrophoresis. The -RT samples serve as controls for the absence of plasmid DNA in the RNA preparations. C: RNA was isolated from UTP_BAD::<i>bamA</i> containing pFP10-Bm<i>bamA</i> (Bmal) or pFP10-Bp<i>bamA</i> (Bper) grown in the presence of arabinose and IPTG and processed as explained for panel B.</p

Expression and assembly of <i>E. coli</i> BamA in <i>N. meningitidis</i>.

<p>Cell envelopes were analyzed by SDS-PAGE followed by immunoblotting using anti-<i>Ec</i>BamA antiserum or by staining with Coomassie Brilliant Blue. BamA is indicated with arrowheads. A: Immunoblots of cell envelopes of uninduced (-) or induced (+) HB-1 cells containing pFP10-Ec<i>bamA</i> carrying <i>bamA</i> under an IPTG-inducible promoter. B: Cell envelopes of HB-1 (Nm) expressing <i>E</i>cBamA or of <i>E. coli</i> strain DH5α (Ec) were extracted with urea and the input (i), urea-insoluble (p) and -soluble (s) fractions were analyzed by SDS-PAGE followed by staining with Coomassie Brilliant Blue (lower panels) or immunoblotting with anti-<i>Ec</i>BamA antiserum (upper panels). Neisserial porins are indicated with asterisks. C: Cell envelopes of <i>E. coli</i> strain DH5α (Ec) or HB-1 (Nm) expressing <i>Ec</i>BamA were treated (+) or not (-) with trypsin for 1 h and analyzed by SDS-PAGE followed by immunoblotting with anti-<i>Ec</i>BamA antiserum. Trypsin-protected <i>Ec</i>BamA fragments in <i>E. coli</i> are indicated with an asterisk. </p

Functionality of BamA variants in <i>E. coli</i>.

<p>Growth of <i>E. coli</i> strain UTP_BAD::<i>bamA</i> carrying no plasmid (panel A) or plasmids encoding the proteins indicated in the upper right hand side of the graphs was assessed by measuring the OD₆₀₀. Strains were grown in the presence of glucose (open circles), arabinose (closed diamonds) or IPTG (closed triangles) in LB at 37°C. Cultures were diluted into fresh medium at the time points indicated by arrows.</p

Schematic representation of the BamA chimeras used in this study.

<p>A: Two sets of chimeras were constructed based on two different topology models for BamA. In one model, the POTRA domains (P1-P5) are connected via a hinge region (H) to a 12-stranded β-barrel (left). In the other model, the POTRA domains are directly connected to a larger, 16-stranded β-barrel (right). <i>E. coli</i>-derived polypeptides are indicated in grey and <i>N. meningitidis</i> derived polypeptides in white. B: ClustalW alignment of partial sequences (residues 417-485, counting the signal sequence) of <i>N. meningitidis</i> and <i>E. coli</i> BamA. The C terminus of POTRA5 (T423) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085799#B18" target="_blank">18</a>] and the N terminus of the predicted 12-stranded β-barrel (V483) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085799#B4" target="_blank">4</a>] are indicated by arrows. The β-strands 1 through 4 plus the start of the fifth, forming the BamA β-barrel as determined in the recently published crystal structure of <i>Ng</i>BamA [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085799#B37" target="_blank">37</a>], are indicated with grey arrows. </p

Expression and assembly of BamA chimeras in <i>N. meningitidis</i>.

<p>A: Derivatives of strain HB-1 containing plasmids encoding the chimeric proteins indicated above the panels were grown with or without IPTG. Cell envelopes were isolated and analyzed by SDS-PAGE and immunoblotting with the antisera shown below the blots. The chimeric proteins are indicated with asterisks. Note that also chromosome-encoded <i>Nm</i>BamA is detected with the α-N-<i>Nm</i>BamA antiserum (indicated with the arrowhead). B: Trypsin sensitivity of BamA variants in <i>N. meningitidis</i> cell envelopes. Cell envelopes of strain HB-1 expressing the indicated BamA variant were treated overnight with or without trypsin and analyzed on immunoblots. Chromosomally encoded <i>Nm</i>BamA in the parent strain HB-1 (wt) yielded three tryptic fragments indicated as I, II and III. Additional digestion products of Ec₄₇₉Nm and Ec₄₂₃Nm are indicated with asterisks. Arrowheads indicate the positions of undigested <i>Nm</i>BamA and the Ec₄₂₃Nm and Ec₄₇₉Nm chimeras, which all have indistinguishable electrophoretic mobilities. In the righ-hand panel, asterisks indicate the positions of undigested Nm₄₂₃Ec and Nm₄₈₀Ec. The blot required a long exposure time to visualize Nm₄₈₀Ec which additionaly revealed a cross-reactive band just below the signal of the chimeras. C: Extractability of BamA variants from cell envelopes with urea. Cell envelopes of <i>N. meningitidis</i> strain HB-1 producing the chimeras indicated at the right were extracted with urea and the input (i), urea-insoluble (p) and -soluble (s) fractions were analyzed by SDS-PAGE and immunoblotting with the antibodies indicated on the left. Arrowheads indicate chromosomally encoded endogenous <i>Nm</i>BamA. In the lower two panels, the positions of endogenous BamA and the chimeric proteins are indistinguishable.</p

Analysis of the β-barrel domain of <i>Nm</i>BamA.

<p>A: Intact HB-1 cells and HB-1 cells producing Ec₄₂₃Nm or Ec₄₇₉Nm were treated with the indicated concentrations of trypsin for 15 min and processed for immunoblotting with α-POTRA1_Nm (left panels) or α-<i>Ec</i>BamA (right panels) antibodies. B. Cell envelopes of HB-1 producing the proteins indicated at the right were treated with urea and processed for immunoblotting with α-C-<i>Nm</i>BamA antiserum. C: Cell envelopes of HB-1 producing the proteins indicated on top were treated overnight with or without trypsin and analyzed on immunoblots with α-C-<i>Nm</i>BamA antiserum. The # signs indicate the positions of the truncated ₄₂₀<i>Nm</i> BamA and ₄₈₁<i>Nm</i>BamA variants, wheras the * signs indicate tryptic fragments derived from the chimeric Ec₄₂₃Nm or Ec₄₇₉Nm proteins (similar to those shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085799#pone-0085799-g006" target="_blank">Fig. 6B</a>).</p

data_sheet_1.docx

<p>Overactivation of the alternative pathway of the complement system is associated with the renal diseases atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy (C3G). C3 nephritic factors (C3NeF) play an important role in C3G pathogenesis by stabilizing the key enzymatic complex of complement, the C3 convertase. However, the reliability of assays detecting these autoantibodies is limited. Therefore, in this study, we validated and optimized a prototype hemolytic method for robust detection and characterization of factors causing convertase overactivity in large patient cohorts. The assay assesses convertase activity directly in the physiological milieu of serum and therefore is not restricted to detection of stabilizing autoantibodies such as C3NeF but may also reveal genetic variants resulting in prolonged convertase activity. We first defined clear cutoff values based on convertase activity in healthy controls. Next, we evaluated 27 C3G patient samples and found 16 positive for prolonged convertase activity, indicating the presence of factors influencing convertase stability. In three patients, the overactive convertase profile was persistent over disease course while in another patient the increased stability normalized in remission. In all these four patients, the convertase-stabilizing activity resided in the purified immunoglobulin (Ig) fraction, demonstrating the autoantibody nature. By contrast, the Igs of a familial aHUS patient carrying the complement factor B mutation p.Lys323Glu did not reveal convertase stabilization. However, in serum prolonged convertase activity was observed and segregated with the mutation in both affected and unaffected family members. In conclusion, we present a robust and reliable method for the detection, characterization, and evaluation over time of factors prolonging convertase activity (C3NeF or certain mutations) in patient cohorts. This assay may provide new insights in disease pathogenesis and may contribute to the development of more personalized treatment strategies.</p

Analysis of Rare Variants in the <i>C3</i> Gene in Patients with Age-Related Macular Degeneration

<div><p>Age-related macular degeneration (AMD) is a progressive retinal disorder affecting over 33 million people worldwide. Genome-wide association studies (GWASs) for AMD identified common variants at 19 loci accounting for 15–65% of the heritability and it has been hypothesized that the missing heritability may be attributed to rare variants with large effect sizes. Common variants in the complement component 3 (<i>C3</i>) gene have been associated with AMD and recently a rare <i>C3</i> variant (Lys155Gln) was identified which exerts a large effect on AMD susceptibility independent of the common variants. To explore whether additional rare variants in the <i>C3</i> gene are associated with AMD, we sequenced all coding exons in 84 unrelated AMD cases. Subsequently, we genotyped all identified variants in 1474 AMD cases and 2258 controls. Additionally, because of the known genetic overlap between AMD and atypical hemolytic uremic syndrome (aHUS), we genotyped two recurrent aHUS-associated <i>C3</i> mutations in the entire cohort. Overall, we identified three rare variants (Lys65Gln (<i>P</i> = 0.04), Arg735Trp (OR = 17.4, 95% CI = 2.2–136; <i>P</i> = 0.0003), and Ser1619Arg (OR = 5.2, 95% CI = 1.0–25; <i>P</i> = 0.05) at the <i>C3</i> locus that are associated with AMD in our EUGENDA cohort. However, the Arg735Trp and Ser1619Arg variants were not found to be associated with AMD in the Rotterdam Study. The Lys65Gln variant was only identified in patients from Nijmegen, the Netherlands, and thus may represent a region-specific AMD risk variant.</p></div