Sequence heterogeneity of PsaA, a 37-kilodalton putative adhesin essential for virulence of Streptococcus pneumoniae

psaA encodes a 37-kDa putative pneumococcal surface adhesin, Although its complete nucleotide sequence has been determined, its contribution to the pathogenicity of Streptococcus pneumoniae has not previously beep assessed, In this study, we used a PCR-amplified internal fragment of the psaA gene from S, pneumoniae type 2 strain D39 cloned in pVA891, to direct the construction of D39 derivatives in which the psaA gene had been specifically interrupted, by insertion-duplication mutagenesis. Two independent D39 psaA mutants (PsaA(1)(-) and PsaA(2)(-)) were significantly less virulent (as judged by intranasal or intraperitoneal challenge of mice) than either the wild-type D39 strain or a derivative of PsaA(1)(-) in which the psaA gene had been reconstituted by back-transformation with an intact copy of the cloned gene, pVA891-directed mutagenesis of an open reading frame (designated ORF3) immediately 3' to psaA or insertion of pVA891 between psaA and ORF3 had no impact on intranasal virulence. However, a small but significant difference in virulence was observed between these two derivatives and the parental D39 strain in a low-dose intraperitoneal challenge model, suggesting that the ORF3 product may also contribute to pathogenesis, Adherence of PsaA(1)(-) to A549 cells (type II pneumocytes) was only 9% of that for D39, while the ORF3-negative strain exhibited intermediate adherence (23%). This is the first functional evidence that PsaA is an adhesin, Sequence analysis of the psaA gene from D39 indicated significant deviation from that previously published for the homolog from S, pneumoniae R36A, The deduced amino acid sequences of mature PsaA from the two strains had only 81% homology, with the bulk of the variation occurring in the amino-terminal portion.