Interaction of phase variation, host and pressure/gas composition: Pneumococcal gene expression of PsaA, SpxB, Ply and LytA in simulated middle ear environments

Abstract

Objective Streptococcus pneumoniae, a leading cause of otitis media (OM), undergoes spontaneous intra-strain variations in colony morphology. Transparent (T) variants are more efficient in colonizing the nasopharynx while opaque (O) variants exhibit greater virulence during systemic infections. This study was intended to delineate the underlying molecular mechanisms by which the predominant S. pneumoniae variant efficiently infects the middle ear (ME) mucosa. Methods Human ME epithelial cells were preconditioned for 24 h under one of the three gas/pressure conditions designed to simulate those for (1) normal ME (NME), (2) ME with Eustachian tube obstruction (ETO) and (3) ME with tympanostomy tube placement (TT), and then were incubated with ∼10 7 CFU/ml of either T or O variants of S. pneumoniae (6A) for 3 h. Relative expression levels of genes encoding virulence factors, PsaA (surface adhesion), SpxB (pyruvate oxidase), Ply (pneumolysin), and LytA (autolysin) were assessed separately in epithelium-attached and supernatant bacteria 3 h post infection using real-time PCR. Results Basal levels of the virulence molecules in inocula were comparable between two variants. However, relative expression levels of the gene transcripts were significantly induced in epithelium-attached T variants 3 h after infection. Comparing with NME and TT conditions, ETO environment produced the largest effect on the differential expression of the virulence genes in the infected ME epithelial cells between T (induced) and O (suppressed) phenotypic pneumococci. Conclusions T variant is a predominant phenotype responsible for the pathogenesis of pneumococcal OM.