Mechanisms regulating the adhesion of S. pneumoniae to activated epithelial cells have been clarified; however, factors involved in attachment to resting epithelial cells have not been elucidated. S. pneumoniae were labeled with [14C] N-acetyl-glucosamine, adhered to confluent monolayers of alveolar type II pulmonary epithelial cells (A549) for 60 min. at 37°C and washed two times prior to trypsin relase of adherent pneumococci. Total adhesion was calculated as [adherent cpm/inoculated cpm]× 100.

Pneumococcal adhesion to resting epithelial cells increased with inoculum size and growth phase, rising significantly during mid- to late exponential phase. Adhesion of the transparent phenotype was greater than the opaque phenotype (28±2% vs. 18±1%, p<.001). An encapsulated serotype 3 strain (6303) exhibited low adhesion (4±4%), while adhesion of its unencapsulated transposon mutant (DW3.8) increased dramatically(25±2%). Subsequent experiments were conducted with 106 cells of strain R6x, an unencapsulated serotype 2 isolate, at mid- to late exponential phase.

Removal of pneumococcal surface proteins with a 2% choline wash increased adhesion of R6x from 24±2% to 31±2% (p=.001). Preincubation of epithelial cells with the choline wash containing surface proteins did not block adhesion; therefore, choline did not remove pneumococcal adhesins. Adhesion of R6x was significantly increased when expression of the C3-degrading proteinase was interrupted by insertional mutagenesis(24±1 vs. 28±1%, p=.01); as predicted, a choline wash of this isogenic mutant did not further increase adhesion. As a control, insertional mutagenesis of PspA in strain WU2 did not increase adhesion.

We propose that pneumococcal adhesins involved in attachment to resting epithelial cells may be masked by pnemococcal surface proteins. Removal of surface proteins with choline or mutation of the C3-degrading proteinase, but not PspA, unmasks adhesins involved in initial attachment to resting epithelial cells.