Involvement of Lsp, a Member of the LraI-Lipoprotein Family in<i>Streptococcus pyogenes</i>, in Eukaryotic Cell Adhesion and Internalization

<jats:title>ABSTRACT</jats:title><jats:p>Three open reading frames (ORFs) were identified by a genome walking strategy in the genomes of serotype M49 group A streptococcal (GAS) strains CS101 and 591. These ORFs were located between the<jats:italic>mga</jats:italic>core regulon and the dipeptide permease operon. The deduced amino acid (aa) sequences contained signature sequences indicative of a lipoprotein (306 aa), an intracellular protein (823 aa), and a secreted peptide (66 aa), respectively. ORF1 (named Lsp for lipoprotein of<jats:italic>Streptococcus pyogenes</jats:italic>) and ORF2 exhibited a high degree of homology to the<jats:italic>lmb</jats:italic>/ORF2 genes of<jats:italic>S. agalactiae</jats:italic>(B. Spellerberg et al., Infect. Immun. 67:871-878, 1999). The three ORFs were found to be present in each of the 27 GAS serotype strains tested. Transcription analysis revealed a polycistronic<jats:italic>lsp</jats:italic>/ORF2 and a monocistronic ORF3 message that were detected primarily at the transition from exponential to stationary growth phase.<jats:italic>lsp</jats:italic>and ORF2 mutants, ORF2- and ORF3-luciferase reporter fusions, and antiserum against recombinant Lsp were produced to examine the biological role of these genes. Although high Zn<jats:sup>2+</jats:sup>and Cu<jats:sup>2+</jats:sup>ion concentrations decreased<jats:italic>lsp</jats:italic>operon expression, Lsp did not transport divalent cations as described for other LraI-type operons. The<jats:italic>lsp</jats:italic>mutant had reduced fibronectin binding. Although no direct binding of Lsp to fibronectin could be demonstrated, the<jats:italic>lsp</jats:italic>mutant showed decreased transcription of<jats:italic>prtF2</jats:italic>encoding the fibronectin-binding protein F2. Both the<jats:italic>lsp</jats:italic>and ORF2 mutants showed decreased laminin binding. Adherence to and internalization into A549 epithelial cells of both mutants was reduced without a detectable effect on eukaryotic cell viability. The transcription of a number of virulence factors was altered in the<jats:italic>lsp</jats:italic>mutants and ORF2 mutants. The changes in laminin binding and eukaryotic cell internalization could be explained by changes in transcription of<jats:italic>speB</jats:italic>(cysteine protease) and/or the global regulators<jats:italic>mga</jats:italic>,<jats:italic>csrRS</jats:italic>, and<jats:italic>nra</jats:italic>.</jats:p>