Schematic representation of Δ<i>idtr</i> construction.

<p>H-<i>HindIII</i>, B-<i>BamHI</i>. T1, T2 amplify the <i>tmp</i> cassette (495 bp); T1 and T2 have H and B at 5′ end. I1, I2 and I3, I4 amplify 5′ and 3′ end of <i>idtr</i>. I2 and I3 have H and B at 5′ end. I1, I2 amplify a 945 bp product and I3, I4 amplify a product of 489 bp.</p

Growth of TIGR4 and Δ<i>idtr</i> and Gram stain morphology of Δ<i>idtr</i> in vitro.

<p>The growth of TIGR4 and Δ<i>idtr</i> in CDM and iron depleted CDM was monitored by measuring absorbance at 600 nm. B) The morphology of Δ<i>idtr</i> was observed in (I) Iron depleted CDM (II) CDM by Gram staining. The results shown are average of three independent experiments cells grown in iron.</p

Survival of mice infected with TIGR4 and Δ<i>idtr</i>.

<p>CBA/CaHN-Btk<i>^xid</i>/J mice were inoculated (A) intranasally with 10⁶ CFU and (B) intravenously with 10⁵ CFU of TIGR4 and Δ<i>idtr</i>. Kaplan Meier curves shown are a representative of triplicate experiments (n = 5 in each experiment).</p

Pneumococcal gene expression in Δ<i>idtr</i> in vitro and in vivo.

<p>Expression of ten pneumococcal genes in Δ<i>idtr</i> relative to TIGR4 in CDM (A) and from nasopharyngeal washes, lung homogenates and blood samples (B) was quantified by RT-PCR. Each experiment was performed using three separate biological sample, each done in triplicate.</p

Average bacterial counts from mouse blood TIGR4 and Δ<i>idtr</i>.

<p>A group of 5 mice each were infected intravenously with 10⁵ CFU of TIGR4 or Δ<i>idtr</i>. Blood samples at different time points were plated to determine bacterial counts. The error bars represent standard error of mean. ^**Significantly decreased as compared to TIGR4 infected blood counts (P<0.01).</p

Ply activity and expression.

<p>Effects of <i>potD</i> deletion on hemolytic activity were assessed via hemolysis assay, in which pneumococcal lysates were incubated with sheep RBCs and lysis was monitored. Lysis by Triton X-114 was set at 100%. Effects on Ply synthesis were determined using a standard indirect ELISA, in which WT values were set at 100%. Deletion of <i>potD</i> from MNZ67 significantly reduced hemolytic potential (A). A significant reduction in Ply production was also observed upon deletion of <i>potD</i> (B). All samples were analyzed in triplicate and experiments were performed at least 3 times. Error bars denote standard error of the mean.</p

Chinchilla acute OM.

<p>Experimental OM was assessed using a chinchilla model. Animals were infected via transbullar injection and were euthanized 4 days post infection. Pneumococci recovered from chinchilla bullae did not significantly differ between WT MNZ67 and PIP01 (A). Significantly less CFU were recovered from chinchillas infected with T4Δ<i>potD</i> in comparison to wild type T4 (B). For each strain, 3 chinchillas were infected in both ears, yielding 6 individual infections. Experiments were performed twice and error bars denote standard error of the mean.</p

Persistence in murine lungs.

<p>The lungs of intratracheally infected mice were collected 2 days post infection, and the ability of MNZ67 and PIP01 to persist in the lungs was determined by enumerating CFU. Significantly more pneumococci were recovered from mice infected with PIP01. Five mice were infected with each strain. Error bars denote standard error of the mean.</p

Epithelial cell adhesion and invasion.

<p>Changes in epithelial cell adhesion and invasion were assessed using adhesion/invasion assays with either Detroit 562 (pharyngeal) or A549 (lung) human epithelial cells. Epithelial cells were incubated with 10⁷ pneumococci and adhered or internalized pneumococci were enumerated on BA. Adhesion to Detroit 562 was not significantly affected by the absence of <i>potD</i> in MNZ67 (A), but adhesion to A549 cells was significantly increased in the absence of <i>potD</i> (B). A significant difference was not observed between MNZ67 and PIP01 when examining Detroit 562 or A549 cell invasion (C and D). All samples were analyzed in triplicate and experiments were performed at least 3 times. Error bars denote standard error of the mean.</p

Differential effects on biofilm production.

<p>Effects of <i>potD</i> deletion on biofilm production were determined using a biofilm assay, in which biofilm formation was assessed 24 hours after seeding 1x10⁵ pneumococci in a 24 well plate. Data is presented as OD₆₃₀. NESp PIP01 formed significantly more biofilm than WT MNZ67 (A), while T4Δ<i>potD</i> produced significantly less biofilm than WT T4 (B). Samples were analyzed in triplicate and experiments were performed at least three times. Error bars denote standard error of the mean.</p