AdcAII of Streptococcus pneumoniae Affects Pneumococcal Invasiveness.

Across bacterial species, metal binding proteins can serve functions in pathogenesis in addition to regulating metal homeostasis. We have compared and contrasted the activities of zinc (Zn2+)-binding lipoproteins AdcA and AdcAII in the Streptococcus pneumoniae TIGR4 background. Exposure to Zn2+-limiting conditions resulted in delayed growth in a strain lacking AdcAII (ΔAdcAII) when compared to wild type bacteria or a mutant lacking AdcA (ΔAdcA). AdcAII failed to interact with the extracellular matrix protein laminin despite homology to laminin-binding proteins of related streptococci. Deletion of AdcA or AdcAII led to significantly increased invasion of A549 human lung epithelial cells and a trend toward increased invasion in vivo. Loss of AdcAII, but not AdcA, was shown to negatively impact early colonization of the nasopharynx. Our findings suggest that expression of AdcAII affects invasiveness of S. pneumoniae in response to available Zn2+ concentrations

Western blot assessing pilus SP0463 expression.

<p>Lysates from T4R, ΔAdcA, and ΔAdcAII strains were blotted and probed with anti-SP0463 antibody and developed. Black arrow indicates SP0463 pilus monomer.</p

Dot blot demonstrating lack of laminin binding by AdcAII.

<p>A) Laminin (2 μg, 5 μg, and 10 μg spots) or anti-AdcAII antibody (1:100, 1:1000, 1:5000 spots) as control were probed with biotinylated recombinant AdcAII protein. B) To demonstrate laminin could interact with ligands using this method laminin protein (2 μg, 5 μg, and 10 μg spots) or anti-laminin receptor antibody (1:100, 1:1000, 1:5000 spots) as a control probed with recombinant laminin receptor and developed with anti-LR antibody.</p

Effects of zinc concentration on growth of Adc mutants.

<p>A) Representative growth curve from three runs of T4R wild type and Adc mutants grown in THY media (pH6.5) in the absence (A) or presence (B) of the Zn-specific chelator TPEN (21μM).</p

Effect of AdcA and AdcAII on adhesion and invasion in A549 cell line.

<p>A) Adhesion relative to T4R (100% representing approximately 0.9% of inoculum adhered). Bacteria were incubated with A549 cells for 30 mins in 24-well plates and adherent bacteria were quantitated. ** = P<0.0005. B) Invasion relative to T4R (100% representing approximately 0.03% of inoculum invaded). Bacteria were incubated for 2 hrs with A549 cells, followed by 1 hr in the presence of antibiotics to kill extracellular bacteria; cells were lysed, and intracellular bacteria were quantitated. * = P<0.005 and ** = P<0.0005. C) Fluorescent images of A549 cells incubated with T4R, ΔAdcA ΔAdcAII, ΔAdcAII+ as in B. Cells were washed, permeabilized, and bacteria were stained with anti-TEPC-15 antibody (in red). Cell nuclei are stained with DAPI (blue). D) Invasion relative to T4R (%). Bacteria were incubated for 2 hrs with A549 cells with 200μM ZnSO₄, followed by 1 hr in the presence of antibiotics to kill extracellular bacteria; cells were lysed, and intracellular bacteria were quantitated. All experiments were conducted at least three times. Each experiment consisted of triplicate sample wells.</p

Effect of AdcAII on <i>in vivo</i> colonization and invasion.

<p>A) For colonization studies, mice were inoculated intranasally with bacteria (7x10³ CFU). At 24 and 48 hrs, mice were sacrificed and 200μL of PBS was passed from the trachea through the nose. Nasal passages were also collected, homogenized, and serial dilutions were plated. B) For invasion studies, mice were inoculated intranasally with bacteria (3.6x10⁴ CFU). Serial dilutions of blood samples were plated at 24 and 48 hrs. Results have been pooled from two independent experiments.</p

Effects of zinc on expression of <i>adcA</i> and <i>adcAII</i> in zinc rich (100μM ZnSO₄) or low zinc environment (30μM TPEN).

<p>A) Expression of <i>adcA</i> (grey) and <i>adcAII</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146785#pone.0146785.ref028" target="_blank">28</a>] in wild type T4R as measured by qRT-PCR ΔΔCT analysis. B) Expression of <i>adcA</i> in ΔAdcAII. C) Expression of a<i>dcAII</i> in ΔAdcA.</p