Streptococcus pneumoniae infection of host epithelial cells via polymeric immunoglobulin receptor transiently induces calcium release from intracellular stores

Abstract

The pneumococcal surface protein C (PspC) is a major adhesin of Streptococcus pneumoniae (pneumococci) that interacts in a human-specific manner with the ectodomain of the human polymeric immunoglobulin receptor (pIgR) produced by respiratory epithelial cells. This interaction promotes bacterial colonization and bacterial internalization by initiating host signal transduction cascades. Here, we examined alterations of intracellular calcium ([Ca(2+)](i)) levels in epithelial cells during host cell infections with pneumococci via the PspC-hpIgR mechanism. The release of [Ca(2+)](i) from intracellular stores in host cells was significantly increased by wild-type pneumococci but not by PspC-deficient pneumococci. The increase in [Ca(2+)](i) was dependent on phospholipase C as pretreatment of cells with a phospholipase C-specific inhibitor U73122 abolished the increase in [Ca(2+)](i). In addition, we demonstrated the effect of [Ca(2+)](i) on pneumococcal internalization by epithelial cells. Uptake of pneumococci was significantly increased after pretreatment of epithelial cells with the cell-permeable calcium chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid-tetraacetoxymethyl ester or use of EGTA as an extracellular Ca(2+)-chelating agent. In contrast, thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+)ATPase, which increases [Ca(2+)](i) in a sustained fashion, significantly reduced pIgR-mediated pneumococcal invasion. Importantly, pneumococcal adherence to pIgR-expressing cells was not altered in the presence of inhibitors as demonstrated by immunofluorescence microscopy. In conclusion, these results demonstrate that pneumococcal infections induce mobilization of [Ca(2+)](i) from intracellular stores. This may constitute a defense response of host cells as the experimental reduction of intracellular calcium levels facilitates pneumococcal internalization by pIgR-expressing cells, whereas elevated calcium levels diminished bacterial internalization by host epithelial cells.

FIGURE 1.

Effect of live pneumococci (A), PFA-fixed pneumococci (B), or latex beads (C) on [Ca⁺²]_i in Calu-3 and MDCK-hpIgR cells. Indo-1/AM-loaded cells were suspended in HEPES-buffered saline in the cuvette of the spectrofluorimeter and treated with or without (control) pneumococci or latex beads at the indicated time (arrow), and the intracellular calcium level was recorded further for 25 min. Traces represent means ± S.D. (S.D. at every full minute for clarity) of n = 5 experiments using different cell preparations. p < 0.05 in A and B when compared with the controls 5 min after adding bacteria but is not significant in C.

FIGURE 2.

Interactions of PspC with hpIgR induces elevations in intracellular calcium levels in host epithelial cells. A and B, levels of intracellular concentration [Ca⁺²]_i in Calu-3 and MDCK-hpIgR following treatment with PspC-deficient live pneumococci (A) and PspC-deficient PFA-fixed pneumococci (B). C and D, effect of anti-PspC antibodies (C) and purified PspC protein fragments PspC-SH12 or PspC-SH3 (D) on intracellular [Ca⁺²]_i. Indo-1/AM-loaded cells were suspended in HEPES-buffered saline in the cuvette of the spectrofluorimeter and treated at the indicated time points (arrows) with isogenic pspC-mutant pneumococci (live or PFA-fixed), PspC proteins, or pneumococci preincubated with antibodies directed against PspC. Untreated cells were used as a control (control). The [Ca⁺²]_i was recorded before treatment and 25 min after addition of bacteria or proteins. E, intracellular calcium levels in MDCK wild-type upon exposure to PFA-fixed wild-type pneumococci or PspC-SH12. Traces represent means ± S.D. (S.D. at very full minute for clarity) of n = 6 experiments using different cell preparations.

FIGURE 3.

Interactions of PspC with hpIgR in Calu-3, and MDCK-hpIgR cells induce calcium release from intracellular stores. Effect of preincubation of Calu-3 and MDCK-hpIgR cells with 2-APB (50 μm) on the levels of [Ca²⁺]_i upon addition of live S. pneumoniae (A), PFA-fixed S. pneumoniae (B), or purified PspC protein derivative PspC-SH12 (C) to indo-1/AM-loaded cells suspended in HEPES-buffered saline in the cuvette of the spectrofluorimeter. Traces represent means ± S.D. (S.D. at very full minute for clarity) of n = 4 experiments using different cell preparations. p < 0.05, when compared with incubations in the absence of 2-APB (control, Ctrl) but in the presence of bacteria or protein.

FIGURE 4.

PspC-mediated increases in intracellular calcium levels in Calu-3 and MDCK-hpIgR cells depend on activation of phospholipase C. Effect of the PLC inhibitor U73122 (10 μm) or its inactive analog U73343 on [Ca⁺²]_i in Calu-3 and MDCK-hpIgR upon addition of live S. pneumoniae (A), PFA-fixed S. pneumoniae (B), PspC protein SH12 (C), or after activation of purinergic receptors with ATP (100 μm) (D). Traces represent means ± S.D. (S.D. at very full minute for clarity) of n = 4 experiments using different cell preparations.

FIGURE 5.

Elevated intracellular calcium concentrations block pneumococcal internalization by Calu-3 and MDCK-hpIgR cells. A, addition of 250 nm thapsigargin to indo-1/AM-loaded Calu-3 and MDCK-hpIgR cells induced elevations in [Ca²⁺]_i due to the activation of capacitative calcium influx. Traces represent means ± S.D. (S.D. at very full minute for clarity) of n = 3 experiments using different cell preparations. B, internalization and intracellular survival of pneumococci in Calu-3 and MDCK-hpIgR cells was determined in the absence (none) or presence of thapsigargin (0.1, 1.0, and 5.0 μm) as determined using the antibiotic protection assay. Pneumococcal invasion in the absence of thapsigargin was set to 100%. Pneumococcal uptake by Calu-3 and MDCK-hpIgR cells was significantly attenuated by pretreatment of epithelial cells with thapsigargin. Data represent means ± S.D. (n = 3). Means of experiments in the presence of thapsigargin were significantly different from those in the untreated controls (*, p < 0.001).

FIGURE 6.

Buffering of intracellular calcium or inhibition of PspC-mediated calcium mobilization facilitates internalization of pneumococci in Calu-3 and MDCK-hpIgR cells. A, indo-1/AM-loaded cells were incubated for 30 min in the absence (none) or in the presence of BAPTA-AM (10 μm) during the recovery phase, and intracellular calcium levels were monitored. [Ca²⁺]_i levels were significantly reduced in BAPTA-AM-pretreated cells compared with the untreated controls (p < 0.01). Traces represent means ± S.D. (S.D. at very full minute for clarity) of n = 6 experiments using different cell preparations. B, effect of preloading Calu-3 and MDCK-hpIgR cells with BAPTA-AM on internalization and intracellular survival of S. pneumoniae after 3 h of infection of epithelial cells as determined by the antibiotic protection assay. Internalization of pneumococci by host cells in the absence of BAPTA-AM was set to 100%. Data represent means ± S.D. (n = 6). Means of experiments in the presence of BAPTA-AM were significantly different from those in the untreated controls (*, p < 0.001). C, inhibition of pneumococcus-induced calcium mobilization by preincubation of Calu-3 and MDCK-hpIgR cells with 2-APB (50 μm) increased the internalization rate of pneumococci as determined by the antibiotic protection assay 3 h post-infection. Pneumococcal uptake in the absence of 2-APB was set to 100%. Means of experiments in the presence of 2-APB were significantly different from those in the untreated controls (*, p < 0.001).

FIGURE 7.

Chelation of extracellular calcium ions reduced basal [Ca²⁺]_i and facilitates internalization of pneumococci in Calu-3 and MDCK-hpIgR cells. A, recording of basal [Ca²⁺]_i before and after addition of EGTA (3 mm) to suspensions of indo-1/AM-loaded cells at the time point indicated. Traces represent means ± S.D. (S.D. at very full minute for clarity) of n = 6 experiments using different cell preparations. B, effect of EGTA on pneumococcal internalization by Calu-3 and MDCK-hpIgR cells. Numbers of intracellularly surviving pneumococci were determined in the absence (none) or presence of EGTA (0.1, 0.5, 1.0, 2.0, and 4.0 mm) 3 h post-exposure. Internalization of pneumococci in the absence of EGTA was set to 100%. Means of experiments in the presence of EGTA were significantly different from those in the untreated controls (*, p < 0.001).

FIGURE 8.

Pneumococcal adherence to Calu-3 and MDCK-hpIgR cells is not altered in the presence of inhibitors but blocked by anti-PspC antibodies. A and B, host cells were exposed to S. pneumoniae serotype 35A (NCTC10319) for 3 h at 37 °C in the absence or presence of indicated inhibitors. After fixation, adherent pneumococci were visualized using a rabbit anti-pneumococcal IgG and Alexa 488-coupled anti-rabbit antibodies in a Zeiss LSM 510 confocal laser scanning microscope (A). Total number of bacteria associated with host cells was determined after removing unbound extracellular bacteria and plating the cells on blood agar plates (n = 3) (B). C, intracellular fate of pneumococci as determined by the enumeration of the intracellular recovered pneumococci. Unbound extracellular bacteria were removed 3 h post-infection, and after killing of extracellular pneumococci by antibiotic (time point 0), the infections were continued for the indicated hours (n = 3). D and E, anti-PspC antibodies block pneumococcal adherence to and invasion into Calu-3 and MDCK-hpIgR cells. Pneumococci were incubated with anti-PspC antibodies (8) for 30 min prior to host cell infections. Adherence was quantified by determination of host cell-associated bacteria, and internalized bacteria were quantified by the antibiotic protection assay. Pneumococci pretreated with anti PspC antigen showed significantly decreased levels of adherence and invasion as compared with the control (none), untreated pneumococci (n = 3) (*, p < 0.001).