1. Volume 102, Issue 6, Pages 827-837 (September 2000)
The Polymeric Immunoglobulin Receptor Translocates Pneumococci across Human Nasopharyngeal Epithelial Cells 
Jing-Ren Zhang, Keith E Mostov, Michael E Lamm, Masanobu Nanno, Shin-ichiro Shimida, Makoto Ohwaki, Elaine Tuomanen 
Cell 
Volume 102, Issue 6, Pages (September 2000)
DOI: /S (00)

2. Figure 1 Identification of hpIgR as a CbpA-Binding Protein
(A) Schematic illustration of CbpA structure and truncate construction. CbpA in serotype 2 contains a C-terminal choline binding domain (CBD) and two N-terminal repetitive regions R1 and R2. Truncated polypeptides (CbpA1–4) are depicted with the beginning and ending of the amino acid residues in the constructs.
(B) Peptide sequence alignment of the N-terminal CbpA repeats R1 and R2. Identical and similar residues are marked as vertical lines and dots, respectively.
(C) Purification of hpIgR by a CbpA-affinity column as detected by binding of streptavidin (left) or an anti-hpIgR serum (middle). The N-terminal peptide sequences of hpIgR and the 120 kDa and 100 kDa proteins are shown on the right. The numbers (1–7) and the letter (b) at the top indicate the column fractions following addition of 100 mM glycine-HCl (pH 2.5) and the unfractionated biotin-labeled Detroit cell extract, respectively. Molecular sizes are in kDa.
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3. Figure 2
Correlation of R6x Adherence and Invasion with Expression of hpIgR in Detroit Cells
(A) Adherence of R6x (left) or the isogenic CbpA− mutant (right) to Detroit cells. Bacteria were incubated with Detroit cells for 1 hr followed by extensive washing. Diff-Quik staining revealed the adherent bacteria (small blue dots) and host cells with large nuclei (purple bodies).
(B) R6x invasion in Detroit cells as visualized by transmission electron microscopy. Bacterial cells (dark bodies) are shown during initial entry at the surface of the host cell (left) or during internalization into the cell (right). Bar is 0.45 μm (left) or 1.2 μm (right).
(C) Effects of loss of CbpA or hpIgR function on R6x invasion in Detroit cells. R6x and the isogenic CbpA mutant R6x (CbpA− were incubated with Detroit cells in 24-well plates. After killing extracellular bacteria with antibiotics, intracellular bacteria were released by detergent lysis of the cells and quantitated by plating the cell lysates on TSA blood plates. For blocking experiments with anti-CbpA serum or anti-hpIgR serum, R6x bacteria or the cell monolayers were treated with a 1:50 dilution of antiserum or normal rabbit serum (NRS) for 30 min before R6x infection. All assays were performed in 4–6 parallel wells at least three independent times. Results of representative experiments are presented as mean + standard deviation.
(D) Comparison of R6x invasion in different human cell lines. In the inset, immunoblots of human cell lysates were detected by using a rabbit anti-hpIgR serum. The hpIgR-expressing MDCK cells (hSC-1) and the MDCK cells transfected with the pCB6 vector only (Neo-1) were used as controls for immunoblot analysis. R6x and the CbpA mutant were used to perform invasion assays with four human cell lines as in (C).
(E) Enhancement of R6x invasion by treating Detroit cells with cytokines. Prior to bacterial infection, Detroit cells were treated with cytokines for 12 hr at 37°C. Blocking experiments with anti-hpIgR (anti-hSC) were performed as in (C).
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4. Figure 3
Enhancement of R6x Adherence and Invasion by Expression of hpIgR in MDCK Cells
(A) Adherence of R6x (left) or the CbpA− mutant (right) to MDCK cells transfected with pCB6 vector alone (Neo-1), pCB6-hpIgR (hSC-1) or pCB6-rpIgR (A4). Adherence was determined as in Figure 2A.
(B) Invasion of MDCK cells by R6x and the CbpA mutant. Invasion was determined as in Figure 2C.
(C) Blocking of R6x invasion of hSC-1 cells by anti-CbpA or anti-hpIgR sera. Antibody blocking experiments were carried out as in Figure 2C.
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5. Figure 4 Uptake of the CbpA-Coated Beads by Detroit Cells
(A) CbpA1-coated latex beads were incubated with Detroit cells cultured on coverslips to examine attachment of the beads to the cell surface by scanning electron microscopy. The attached beads are surrounded by the cell surface adhesion structures. Bar is 0.8 μm.
(B) Detroit cells internalized CbpA1-coated beads. Internalized beads (shown as six intracellular spheres) were detected by transmission electron microscopy of ultrathin sections. The white areas within the beads arise by chemical damage of processing reagents as shown previously (Lecuit et al. 1997). Bar is 1.6 μm.
(C) CbpA1- or BSA-coated beads were incubated with Detroit cells grown on chamber slides for 1 hr. Unbound beads were removed by extensive washing. The beads attached to the cells were quantitated by using fluorescence microscopy.
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6. Figure 5 Binding of the N-Terminal CbpA Repeats to hpIgR
(A) Precipitation of hpIgR by CbpA-coated beads. Latex beads covalently coated with 4 nmol/109 beads of CbpA truncates (CbpA1–4) were incubated with the cell lysates of Detroit cells (DL) or MDCK cells (hSC-1 and Neo-1). Following removal of unbound proteins by extensive washing, proteins bound to the beads were eluted and separated in an SDS-PAGE gel and immunoblotting was performed using a rabbit anti-hpIgR serum. Beads coated with the same amount of BSA or recombinant VncS were processed in a similar manner. Molecular sizes are in kDa.
(B) Blocking R6x invasion by CbpA polypeptides. Detroit cells were incubated in the absence (None) or presence of 0.2 μM CbpA polypeptides for 30 min before R6x infection. The recombinant VncS (0.2 μM) was used as a negative control. Intracellular bacteria were measured by the invasion assay as in Figure 2C.
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7. Figure 6 Binding of SC to CbpA
(A) CbpA1-coated beads were incubated with human free SC (hSC), SC treated with sodium periodate (hSC-sp), human serum albumin (HSA), or HSA treated with sodium periodate (HSA-sp). BSA-coated beads were incubated with SC as a control (right lane, left panel). Immunoblots of human SC (left lane, left panel) and the precipitated materials were reacted with antiserum against human SC (left and right panels) or HSA (middle panel).
(B) R6x bacteria were preincubated with 10 μg/ml human free SC (hSC), SC treated with sodium periodate (hSC-sp), HSA, or HSA treated with sodium periodate (HSA-sp) for 30 min at 37°C before addition to Detroit cells. Intracellular bacteria were measured by the invasion assay as in Figure 2C.
(C) Before R6x infection, Detroit cells were incubated at 37°C for 30 min in the absence (None) or presence of 2 μM PAF receptor antagonist (L123), 2 μM inactive isomer of the antagonist (L124), control rabbit serum (NRS), rabbit antiserum against hpIgR (α-hSC), or rabbit antiserum against the human PAF receptor (α-PAFr). Serum samples were used at a final dilution of 1:50. Bacterial invasion was quantitated as above.
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8. Figure 7 Transmigration and Colonization of the Pneumococcus
(A) Availability of hpIgR on the apical surface of the fully polarized epithelial cells. MDCK cells transfected with pCB6 vector only (Neo-1) or with the hpIgR cDNA (hSC-1) were allowed to form fully polarized monolayers on 12 mm Transwell membranes (0.2 μm pore size) before infection with R6x or the CbpA mutant R6x (CbpA−). To test the blocking effects of the antiserum against hpIgR on R6x invasion, a 1:50 dilution (250 μl) of the rabbit antiserum (anti-hSC) or control serum (NRS) was incubated with the cell monolayer at 37°C for 30 min before addition of 250 μl R6x suspension (107 bacteria). The invasion experiments were carried out as in Figure 2C.
(B) Transmigration of R6x across human nasopharyngeal monolayers. Detroit cells grown on Transwell membranes were infected with a 1:1 mixture of R6x and the CbpA mutant in the upper chamber. Bacteria in the lower chambers were quantitated by plating fractions of the medium on TSA blood plates with (selecting the CbpA mutant) or without erythromycin (total bacteria). The specific role of hpIgR in R6x transmigration was assessed by treating the monolayers with a 1:50 dilution of the rabbit antiserum against hpIgR (anti-hSC) or the control serum (NRS) for 1 hr before R6x addition.
(C) Decreased nasal colonization of S. pneumoniae in pIgR KO mice. Five pIgR KO and wild-type mice were given 105 CFU of serotype 4 pneumococcus. The nasal bacteria loads in individual animals were determined by nasal washing at 12 and 52 hr post inoculation. The results represent one of two experiments performed at different times.
(D) Delayed onset of pneumococcal bacteremia in p62yes KO mice. Five p62yes KO and control mice were infected intranasally with 105 CFU of serotype 4 pneumococcus. Presented are the levels of bacteremia in individual p62yes KO (open circles) and control mice (filled triangles) at 12 and 28 hr post inoculation. The results represent one of two experiments performed at different times. Statistical analysis as determined by the Mann-Whitney rank sum test supports significantly different onset levels of bacteremia at 28 hr post inoculation between p62yes KO and control mice (P = 0.008).
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