Significant knockdown of MUC1 and MUC16 proteins in both cell lysates and on apical cell surfaces following transfection with vectors expressing shMUC1 or shMUC16 sequences.

<p>(A) Western blots demonstrating that <b>MUC1 protein</b> is lower in both cell lysates (upper left) and on apical cell surfaces (lower left) of cell cultures transfected with shMUC1 containing vectors (shMUC1) compared to the non-transfected control (NT), scrambled shRNA (scr1) controls, as well as with shMUC16 containing vector (shMUC16) or its scrambled shRNA control (scr16). Alleles of MUC1 often differ in size and as they are co-dominantly expressed, two distinct protein sizes are evident on western blots. The graphs to the right of each blot, show densitometric analyses of bands demonstrating that MUC1 protein levels are significantly reduced by 71% in the cell lysates and 60% on apical surfaces relative to NT and scr1 controls and that MUC1 protein levels are not significantly reduced by knockdown of MUC16 (shMUC16) or its scrambled shRNA control (scr16). (B) Similarly, on the left are representative Western blots demonstrating that MUC16 protein levels are lower in cell lysates and biotinylated apical cell surface protein isolates of cells transfected with shMUC16 containing vectors compared to non-transfected (NT), or those transfected with scrambled shRNA for either MUC1 or MUC16 (scr1 and scr16) or shMUC1 containing vectors. The graphs on the right show densitometric analyses of blots indicating that MUC16 protein levels are significantly reduced in cell lysates by 70% and on apical surfaces by 51% in cells transfected with shMUC16 containing vectors in comparison to NT and scr16 controls. For both (A) and (B) protein samples from cell lysates were loaded based on equivalent micrograms of protein, and for cell surface proteins on equivalent cm² of cell growth area. Graphic representation of the relative amounts of MUC1 (upper right) and MUC16 (lower right) was derived through densitometric analyses of the blots, cell lysates were normalized to GAPDH, and all data were expressed relative to the non-transfected control (NT). Significant if p<0.01, (**). ns = non-significant, n = 5–10.</p

Diagram of the distribution of the MAMs MUC1 and MUC16 in the epithelial glycocalyx and their molecular domains.

<p>(A) Electron micrograph showing diagrammatically, the distribution of MUC1 (red) and MUC16 (yellow) within the electron dense glycocalyx (top arrow) present at the tips of membrane folds or microplicae of an epithelial cell. Note the actin filaments inserting into the membrane at the tips of the microplicae where the cytoplasmic tails of the membrane mucins are present (bottom arrow). (B) Both MUC1 and MUC16 have a short cytoplasmic tail, a transmembrane domain and an extended, highly glycosylated extracellular domain that contains tandem repeats of amino acids, rich in serine and threonine, that allow the heavy O-glycosyation of the molecules. MUC1 has one sea urchin sperm protein, enterokinase and agrin (SEA) module, whereas MUC16 has multiple SEA modules interspersed within tandem repeats and, in addition, a shorter cytoplasmic tail and an ERM binding domain. Note that the MUC16 ectodomain is approximately 20 times longer than that of MUC1. It has been estimated that MUC16 can extend up to 250–300 nm into the glycocalyx <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100393#pone.0100393-Gniewek1" target="_blank">[43]</a>. (Electron micrograph taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100393#pone.0100393-Nichols1" target="_blank">[50]</a> with permission.) Scale Bar = 500 nm.</p

Knockdown of MUC16 results in decreased tight junction function and ZO-1/occludin expression, whereas knockdown of MUC1 has no effect on tight junctions.

<p>(A) Immunofluorescence analysis of occludin localization demonstrated normal linear distribution of occludin in the MUC16 scrambled control (scr16) cells (A) as compared to the disrupted localization seen in the shMUC16 cells (B). (C) A highly significant decrease in transepithelial electrical resistance (TER) was observed in the MUC16 knockdown (shMUC16) cell cultures compared to control cultures and shMUC1 cultures. No difference was seen in TER in the MUC1 knockdown (shMUC1) cells n = 15–30. (D) Analysis of the relative mRNA expression of two tight junction genes (ZO-1, occludin) by qPCR demonstrated a significant reduction in their message in the shMUC16 cells compared to the non-transfected (NT), or scrambled shRNA controls (scr1, scr16) and shMUC1 cells. n = 7, **p<0.01, ns = not significant.</p

Knockdown of MUC16 results in an increase in apical cell surface area compared to knockdown of MUC1.

<p>Cell perimeters were labeled with antibodies to occludin followed by labeling with FITC conjugated secondary antibodies (A–E). Note the disruptions in the linear localization around the cell peripheries in the MUC16 knockdown cells, shMUC16 (D) compared to the continuous linear localization in the scrambled shRNA controls scr1 (A), scr16 (C) and non-transfected NT (E) controls as well as the MUC1 knockdown shMUC1 cells (B). (F) Measurement of apical cell surface area in the ZO-1 labeled cultures revealed that the mean apical surface area of the shMUC16 cells is significantly larger than those of the NT, scr1, scr16 and shMUC1 cells, all of which have comparable apical cell surface areas. Scale bar = 30 µm. **p<0.01, ns = not significant, n = 7, 5 images/sample.</p

Knockdown of MUC16 results in disruption of the actin cytoskeleton associated with tight junctions and reduces surface microplicae.

<p>Epithelial cultures of non-transfected controls (A) and those transfected with shMUC16 (B) were double labeled with an antibody to occludin (green) and Phalloidin (red) to localize filamentous actin; note the actin filaments associated with the linear occludin antibody binding in A, and the lack of filamentous actin along the disrupted occludin antibody binding in B. Scanning electron micrographs of control epithelial cultures (C), shMUC16 cultures (D) and native epithelium (E). Note fewer prominent microplicae in the cells knocked down for MUC16 in D and also in the larger darker cell of native epithelium (E), that were shown (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100393#pone-0100393-g002" target="_blank">Fig. 2F</a>) to bind less antibody to MUC16. The larger darker cells show fewer microplicae than neighboring smaller light cells. Scale bars = 15 µm in A, B, 10 µm in C, D, 5 µm in E.</p

Knockdown of MUC16 increases bacterial adherence and invasion compared to knockdown of MUC1.

<p>Three types of experiments demonstrate that knockdown of MUC16 increases bacterial adherence and invasion compared to controls, and that knockdown of MUC1 enhances the barrier to bacterial adherence and invasion. In the first experiment, epithelial cultures were incubated with FITC-labeled <i>S. aureus</i> and number of adherent bacteria were counted in ImageJ. Representative images of <i>S. aureus</i> adherent to cultures are shown in (A) scrambled MUC1 shRNA control (scr1), (B) MUC1 knockdown (shMUC1), (C) scrambled shMUC16 control (scr16), (D) MUC16 knockdown (shMUC16) and (E) non-transfected control (NT). Note abundance of adherent bacteria in the MUC16 knockdown cells in image D. (F) Graph illustrating the number of FITC-labeled bacteria adherent to the epithelial cell cultures. Note that knockdown of MUC16 significantly increases adherence of <i>S aureus,</i> whereas knockdown of MUC1 significantly decreases bacterial adherence. In a second type of experiment, (G) the differences in bacterial adherence between the HCLE shMUC1 and HCLEshMUC16 and control cells were corroborated through enumeration of colony-forming units (cfus) of live bacteria recovered after the 1-h incubation with <i>S. aureus.</i> In a third experiment, (H) number of intracellular <i>S. aureus</i> that invaded the cell cultures were counted after incubation of the cultures for 4 h and determining cfus of live bacteria recovered from cell lysates following antibiotic treatment to kill surface bacteria. The three different assays demonstrate that knockdown of MUC16 is associated with a significant increase in bacterial adherence and invasion, and that knockdown of MUC1 does not increase bacterial adherence or invasion, rather the barrier to bacteria is increased. Scale bar = 30 µm. **p<0.01. n = 6–8.</p

Knockdown of MUC16 enhances dye penetrance compared to knockdown of MUC1.

<p>Representative images of cultures of human corneal epithelial cells stably transfected with (A) scrambled shRNA for MUC1 (scr1), (B) shRNA for MUC1 (shMUC1), (C) scrambled shRNA for MUC16 (scr16), (D) shRNA for MUC16 (shMUC16) or the non-transfected control (NT) (E) and then incubated with rose bengal dye to determine the area of the culture that is protected from dye penetrance, an indication of a functional apical glycocalyx barrier. Rose bengal dye is excluded from islands of cells in cultures of non-transfected (NT) and scrambled shRNA controls (scr1, scr16), as well as the MUC1 knockdown cells (shMUC1) cultures. Cells knocked down for MUC16 (shMUC16) do not show as many islands of dye exclusion, indicating increased penetrance of the dye. (F) Quantitative image analyses of the area protected from dye penetrance in each cell type demonstrate a significant decrease in area protected from dye penetrance in the MUC16 knockdown cells. Conversely, there is a significant increase in the area protected from dye penetrance in the MUC1 knockdown (shMUC1) cells. Scale bar = 50 µm. **p<0.01, n = 8.</p

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

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