NheA binding to NheB/C adjusted for different ratios and assayed by flow-cytometry.

<p>(A) Expemplified overlay histogramm of Vero cells incubated with a NheB/C ratio of 10:1 (green histogram) and 1:1 (grey histogramm) followed by addition of 1G4 neutralized rNheA. The artificial adjustment of NheB/C ratio to equal levels will result in the formation of NheB/C heterodimers that recruit less NheA compared to the 10:1 ratio. Cells incubated with neutralized rNheA only are depicted in the white histogramm. (B) bar charts generated according to the flow cytometry experiments with different NheB/C ratios; the colours of the bars correspond to the set-ups in (A). Error bars represent the SD of triplicates.</p

Schematic illustration of the refined pore-forming action of Nhe as substantiated in the present manuscript.

<p>(A) In solution NheA is present as single soluble molecules whereas NheB and -C form complexes as published [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165135#pone.0165135.ref020" target="_blank">20</a>]; (B) next the NheB/C complexes attach to the cell membrane where small permeable pro-pores are formed [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165135#pone.0165135.ref026" target="_blank">26</a>]; the pro-pore formation occurs independent of -A which is still in solution; (C) the penultimate step is the attachment of the A-component to the pro-pore followed by a conformational alteration (D) that leads to full pore-formation and destruction of the target-cell (E).</p

Reactivity of the recombinant NheA fragments in different EIA set-ups substantiate the epitope-mapping experiments based on synthetic peptides and allow the allocation of the 1G4 and 2G11 epitope.

<p>Reactivity of the recombinant NheA fragments in different EIA set-ups substantiate the epitope-mapping experiments based on synthetic peptides and allow the allocation of the 1G4 and 2G11 epitope.</p

Immunofluorescence microscopy of NheA on Vero cells.

<p>Staining of cell-bound NheA after treatment of Vero cells with <i>B</i>. <i>cereus</i> supernatants. (A) MHI 1507 NheB (100 ng ml^-1 and approx. 10 ng ml^-1 NheC) with mAb 1G4 neutralized NheA untreated Vero cells stained with primary and secondary antibody. (B) MHI 1761 (containing NheB at 100 ng ml^-1 and approx. 10 ng ml^-1 NheC) supplemented with neutralized rNheA (200 ng ml^-1). (C) Stained Vero cells treated with rNheA only. (D) Untreated Vero cells stained with primary and secondary antibody. All slides were counterstained with DAPI.</p

Immunofluorescence microscopy of NheA on Vero cells.

<p>Staining of cell-bound NheA after treatment of Vero cells with <i>B</i>. <i>cereus</i> supernatants. (A) MHI 1507 NheB (100 ng ml^-1 and approx. 10 ng ml^-1 NheC) with mAb 1G4 neutralized NheA untreated Vero cells stained with primary and secondary antibody. (B) MHI 1761 (containing NheB at 100 ng ml^-1 and approx. 10 ng ml^-1 NheC) supplemented with neutralized rNheA (200 ng ml^-1). (C) Stained Vero cells treated with rNheA only. (D) Untreated Vero cells stained with primary and secondary antibody. All slides were counterstained with DAPI.</p

Results of the neutralization assays.

<p>(A) Neutralization of toxic activity in supernatants from MHI 1507 by mAb 1A8 (open triangle), mAb 2G11 (black triangle) and mAb 1G4 (open dot). A high reciprocal dilution at the 50% toxic dose indicates a low or absent neutralizing capacity of the antibody applied. The neutralizing effect of 1G4 and 2G11 are similar while 1A8 and the isotype control (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165135#pone.0165135.s002" target="_blank">S2 Fig</a>) are not able to neutralize the cytotoxicity. (B) Consecutive neutralization of serially diluted antibody treated rNheA (applied to NheB/C (MHI 1761) primed cells. With mAb 1A8 or the isotype control the majority of the cells is dead. Error bars represent the SD of triplicates.</p

Reactivity of NheA-specific antibodies 1G4, 1A8 and 2G11 in a Western-blot assay.

<p>Protein lysates applied were as follows: rNheA (1) supernatant of MHI 241 (2) supernatant of MHI 241 concentrated (3). The concentration was performed on Amicon^® (Merck Millipore, Germany) Ultra centrifugal filters with a size exclusion of 30 kDa. The upper band in lane 1 represents rNheA with the 13 kDa thioredoxin tag still attached.</p

Overview on the most important characteristics of the three mAbs against NheA applied in the present study.

<p>Overview on the most important characteristics of the three mAbs against NheA applied in the present study.</p

Neutralization effects assayed by flow-cytometry.

<p>(A) and (B) show the different neutralization capacities of mAb 1G4 and 2G11. (A) supernatant of MHI 241 was neutralized by mAb 1G4 (green histogram) or mAb 2G11 (grey histogramm. (B) NheBC was incubated with 1G4 (green histogram) or 2G11 (grey histogram) neutralized rNheA. White histograms represent the negative controls (Vero cells incubated with primary and secondary antibodies. (C) overlay histogram after incubation of Vero cells with NheBC (adjusted to NheB at 150, 75 and 50 ng ml^-1 and approx.15, 7.5 and 5 ng/ml^-1 NheC, respectively) together with a constant amount of rNheA (150 ng ml^-1) neutralized by mab 1G4; white = negative control; light green = NheB—50 ng/ml; dark green = NheB 150 ng/ml for reasons of overview results obtained with 75 ng/ml^-1 NheB have only be included in the bar chart in (D); (D) means and SDs for NheA cells positive in fluorescence channel 1; colors correspond to those in the overlay histogram (C).</p

Results of the mAb 1G4-based IAC purification of NheA from <i>B</i>. <i>cereus</i> MHI 241 supernatants.

<p>Results of the mAb 1G4-based IAC purification of NheA from <i>B</i>. <i>cereus</i> MHI 241 supernatants.</p