Rose bengal penetrance into HCLE cells increases after SP168 growth culture filtrate treatment.

<p>Brightfield micrographs were taken of HCLE cells stained with rose bengal dye following exposure to (<b>A</b>) medium only control or (<b>B</b>) ZmpC-containing growth culture filtrate obtained from strain SP168. Areas of cells with dye penetrance (p = 0.01, Mann-Whitney test, n = 55) were quantified using ImageJ software (<b>C</b>). Scale bar = 100 µm. These data indicate that the barrier efficiency of epithelial cells after exposure to ZmpC decreases due to loss of MUC16 ectodomain. Increased dye penetrance has been shown previously with siRNA knockdown of MUC16 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032418#pone.0032418-Blalock1" target="_blank">[3]</a>.</p

Isolation of the MUC16 sheddase and its identification as ZmpC.

<p>MUC16 sheddase-enriched fractions were isolated first by DEAE chromatography, with concentrations of sodium chloride eluent shown in the figure on the right (<b>A</b>), then by size exclusion chromatography (<b>B</b>). A sample from fraction 9 (indicated by an arrow) in B was separated by SDS-PAGE and stained using GelCode Blue stain. The 3 bands observed at ∼180 kDa, ∼170 kDa and ∼50 kDa were analyzed by mass spectrometry. Numbers on the left of the gel indicate molecular weight standards in kDa. The Y axes of graphs in (A) and (B) represent the net intensities corresponding to MUC16 signal on western blots. Constitutive MUC16 ectodomain release was observed in fractions whose net intensities correspond to that of the control. The mechanism(s) associated with constitutive MUC16 ectodomain shedding remains unknown.</p

Apical cell surface abundance of MUC16 is reduced following treatment of epithelia with SP168 growth culture filtrate.

<p>Apical membrane levels of MUC16 were determined by cell surface biotinylation. <b>A</b>) Western blot analyses (using M11 antibody) of the amount of released and residual biotin-labeled surface MUC16 after exposure to medium only control or SP168 growth culture filtrate. <b>B</b>) Quantitative analyses of the western blot in (A) showing a reduction in the abundance of surface MUC16 following treatment with the SP168 culture filtrate (p<0.0001, Student's t-test, n = 3). Data for MUC16 surface abundance is expressed as a percentage of the total released plus surface MUC16 ± SEM.</p

MUC16 released from epithelial surfaces by SP168 does not bind to a MUC16 cytoplasmic tail antibody indicating ectodomain release.

<p>HCLE cells were cultured with growth culture filtrate obtained from strain SP168 for 1 hour (SP168), and the resultant media was diluted 1∶3 with DMEM/F12, and subjected to a 10 kDa cutoff concentrator. HCLE cell lysate with intact full-length MUC16 (75 µg) was used as a positive control. Western blot of the resulting concentrate and cell lysate using: <b>A</b>) MUC16CT antibody that recognizes full-length MUC16 at >250 kDa and the cytoplasmic tail between 25–37 kDa, or <b>B</b>) MUC16 extracellular domain (ECD) antibody (M11). The MUC16CT antibody binds to the positive control cell lysate only (arrow), but not to the cell culture supernatants after exposure to SP168 (A). Conversely, M11 antibody binds to a band of appropriate molecular weight in culture supernatants as well as the cell lysate (B). These data indicate that the sheddase releases the MUC16 ectodomain into the cell culture media. Molecular weight standards are on the left in kDa.</p

<i>zmpC</i> is present only in certain strains of <i>S. pneumoniae</i>.

<p>Genomic DNA isolated from all strains of <i>S. pneumoniae</i> under study was subjected to polymerase chain reaction to screen for the <i>zmpC</i> gene. Amplified products were separated on a 1% agarose/TBE gel. Molecular weight markers are represented in kilobase pairs (kbp). Products corresponding to the bands observed at ∼5 kbp were sequenced at the University of Maine DNA Sequencing Facility and identified as <i>zmpC</i>. Data indicated that the <i>zmpC</i> gene was present in <i>S. pneumoniae</i> strain SP168 and serotype 11A, the only strains exhibiting MUC16 sheddase activity. <i>zmpC</i> from <i>S. pneumoniae</i> strain SP168 was found to be 723 bp shorter than that of serotype 11A. The two sequences are 86.5% identical.</p

Epithelial MUC16 ectodomain release is induced by growth culture filtrate from SP168 but not from the SP168 <i>zmpCΔ</i> mutant.

<p><b>A</b>) Western blots using M11 antibody comparing the amount of shed MUC16 in cell culture supernatants recovered after exposure of stratified HCLE and TrBr cells to bacterial growth culture filtrates from strains R6, wild type SP168 and the SP168 <i>zmpCΔ</i> mutant. <b>B</b>) Quantitative analyses of western blots in (A) show that the filtrate derived from the SP168 <i>zmpCΔ</i> mutant is unable to induce MUC16 ectodomain shedding in HCLE cells as well as in TrBr cells (n = 3). All data are represented as MUC16 units relative to the standard ± SEM.</p

<i>S. pneumonia</i>e strain SP168 invades epithelial cells more efficiently than its isogenic <i>zmpC</i>

<p>Δ<b> mutant.</b> HCLE cells were incubated with SP168 and <i>zmpC</i>Δ mutant bacteria for 4 hours. Surface-bound bacteria were killed using an antibiotic protection assay. Quantitation of number of internalized bacteria was performed by serial dilutions of lysed cells and back-plating on blood agar plates. Data are expressed as a percentage of the number of bacteria recovered after the assay, divided by the initial inoculum number ± SEM (p<0.0001, unpaired t-test, n = 6).</p

Diagram of the mechanism by which <i>S. pneumoniae</i> strain SP168 gains entry into epithelial cells.

<p>Scanning electron micrograph of <i>S. pneumoniae</i> strain SP168 (<b>A</b>), which secretes a zinc metalloproteinase, ZmpC, onto the apical surface of the epithelia (<b>B</b>), which has a glycocalyx rich in MUC16 transmembrane mucins that emanate from surface microridges (adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032418#pone.0032418-Gipson3" target="_blank">[47]</a>). The secreted ZmpC induces MUC16 ectodomain release, causing a decrease in amount of MUC16 on the apical surface of the epithelium (<b>C</b>), that compromises the glycocalyx barrier integrity. <i>S. pneumoniae</i>, depicted in orange, is thus able to invade the epithelial cells due to the loss of MUC16 (<b>D</b>).</p

Epithelial MUC16 release by growth culture filtrates from different <i>S. pneumoniae</i> species and <i>S. aureus</i>.

<p><b>A</b>) Western blots using M11, a MUC16 ectodomain-specific antibody, on culture supernatants of HCLE, TrBr, and HCjE cells after exposure to growth culture filtrates from <i>S. pneumoniae</i> strains R6 and SP168, and serotypes 1, 3, 8, and 11A. The 250 kDa standard marker is indicated on the left. <b>B</b>) Quantitative analyses of the western blots in (A) demonstrated that bacterial culture filtrates from only SP168 and serotype 11A induced release of the MUC16 ectodomain (n = 3). <b>C</b>) Western blot with the M11 antibody, on culture supernatants of HCLE cells after exposure to growth culture filtrates from <i>S. pneumoniae</i> strain SP168 and <i>S. aureus</i> ALC1435. <b>D</b>) Quantitative analysis of the western blot in (C) shows the inability of growth culture filtrate derived from <i>S. aureus</i> to induce MUC16 ectodomain shedding (n = 3). Data represent MUC16 units relative to the standard ± standard error of the mean (SEM). <b>E</b>) Western blot using the M11 antibody on known, increasing amounts of a partially purified MUC16 isolate. <b>F</b>) A standard curve that was generated by plotting band intensities that were in the linear range (E) vs known amounts of partially purified MUC16 protein.</p