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

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

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

PspK analysis.

<p>PspK was quantified in pneumococcal lysates by an indirect ELISA, and <i>pspK</i> expression was determined via RT-qPCR. Surface-expressed PspK was assessed by flow cytometry. Significantly more PspK was produced in <i>potD</i> mutant PIP01 when compared to wild type MNZ67 (A). Additionally, significantly higher <i>pspK</i> expression was seen in PIP01 (B). Although increased PspK was observed, no difference was seen in surface associated PspK (C). The ELISA was performed at least three times, and each sample was analyzed in triplicate. RT-qPCR was performed three independent times and data was analyzed using the ΔΔCT method. Flow cytometry was performed twice. Error bars denote standard error of the mean.</p

OM tympanic pathology scores and biofilm scores 4 days post infection.

<p>OM tympanic pathology scores and biofilm scores 4 days post infection.</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

Description of strains used for the studies described in this manuscript.

<p>Description of strains used for the studies described in this manuscript.</p

Names and sequences of primers used in these studies.

<p>Names and sequences of primers used in these studies.</p

Polyamine transporter potABCD is required for virulence of encapsulated but not nonencapsulated <i>Streptococcus pneumoniae</i>

<div><p><i>Streptococcus pneumoniae</i> is commonly found in the human nasopharynx and is the causative agent of multiple diseases. Since invasive pneumococcal infections are associated with encapsulated pneumococci, the capsular polysaccharide is the target of licensed pneumococcal vaccines. However, there is an increasing distribution of non-vaccine serotypes, as well as nonencapsulated <i>S</i>. <i>pneumoniae</i> (NESp). Both encapsulated and nonencapsulated pneumococci possess the polyamine oligo-transport operon (<i>potABCD</i>). Previous research has shown inactivation of the pot operon in encapsulated pneumococci alters protein expression and leads to a significant reduction in pneumococcal murine colonization, but the role of the pot operon in NESp is unknown. Here, we demonstrate deletion of <i>potD</i> from the NESp NCC1 strain MNZ67 does impact expression of the key proteins pneumolysin and PspK, but it does not inhibit murine colonization. Additionally, we show the absence of <i>potD</i> significantly increases biofilm production, both in vitro and in vivo. In a chinchilla model of otitis media (OM), the absence of <i>potD</i> does not significantly affect MNZ67 virulence, but it does significantly reduce the pathogenesis of the virulent encapsulated strain TIGR4 (serotype 4). Deletion of <i>potD</i> also significantly reduced persistence of TIGR4 in the lungs but increased persistence of PIP01 in the lungs. We conclude the pot operon is important for the regulation of protein expression and biofilm formation in both encapsulated and NCC1 nonencapsulated <i>Streptococcus pneumoniae</i>. However, in contrast to encapsulated pneumococcal strains, polyamine acquisition via the pot operon is not required for MNZ67 murine colonization, persistence in the lungs, or full virulence in a model of OM. Therefore, NESp virulence regulation needs to be further established to identify potential NESp therapeutic targets.</p></div