Absence of \u3ci\u3eStreptococcus pneumoniae \u3c/i\u3e Capsule Increases Bacterial Binding, Perisstence, and Inflammation In Corneal Infection

The role of the pneumococcal polysaccharide capsule is largely unclear for Streptococcus pneumoniae keratitis, an ocular inflammatory disease that develops as a result of bacterial infection of the cornea. In this study, capsule-deficient strains were compared to isogenic parent strains in their ability to adhere to human corneal epithelial cells. One isogenic pair was further used in topical ocular infection of mice to assess the contribution of the capsule to keratitis. The results showed that non-encapsulated pneumococci were significantly more adherent to cells, persisted in significantly higher numbers on mouse corneas in vivo, and caused significant increases in murine ocular IL9, IL10, IL12-p70, MIG, and MIP-1-gamma compared to encapsulated S. pneumoniae. These findings indicate that the bacterial capsule impedes virulence and the absence of capsule impacts inflammation following corneal infection

The cholesterol-dependent cytolysin pneumolysin from Streptococcus pneumoniae binds to lipid raft microdomains in human corneal epithelial cells.

Streptococcus pneumoniae (pneumococcus) is an opportunistic bacterial pathogen responsible for causing several human diseases including pneumonia, meningitis, and otitis media. Pneumococcus is also a major cause of human ocular infections and is commonly isolated in cases of bacterial keratitis, an infection of the cornea. The ocular pathology that occurs during pneumococcal keratitis is partly due to the actions of pneumolysin (Ply), a cholesterol-dependent cytolysin produced by pneumococcus. The lytic mechanism of Ply is a three step process beginning with surface binding to cholesterol. Multiple Ply monomers then oligomerize to form a prepore. The prepore then undergoes a conformational change that creates a large pore in the host cell membrane, resulting in cell lysis. We engineered a collection of single amino acid substitution mutants at residues (A370, A406, W433, and L460) that are crucial to the progression of the lytic mechanism and determined the effects that these mutations had on lytic function. Both Ply(WT) and the mutant Ply molecules (Ply(A370G), Ply(A370E), Ply(A406G), Ply(A406E), Ply(W433G), Ply(W433E), Ply(W433F), Ply(L460G), and Ply(L460E)) were able to bind to the surface of human corneal epithelial cells (HCECs) with similar efficiency. Additionally, Ply(WT) localized to cholesterol-rich microdomains on the HCEC surface, however, only one mutant (Ply(A370G)) was able to duplicate this behavior. Four of the 9 mutant Ply molecules (Ply(A370E), Ply(W433G), Ply(W433E), and Ply(L460E)) were deficient in oligomer formation. Lastly, all of the mutant Ply molecules, except Ply(A370G), exhibited significantly impaired lytic activity on HCECs. The other 8 mutants all experienced a reduction in lytic activity, but 4 of the 8 retained the ability to oligomerize. A thorough understanding of the molecular interactions that occur between Ply and the target cell, could lead to targeted treatments aimed to reduce the pathology observed during pneumococcal keratitis

Ply Structure and Sequence Alignment.

<p>A) Ribbon diagram of Ply with the domain 4 loop residues shown at the base. B) The bottom view of domain 4 showing the relative placement of each loop. C) Zoomed in view of domain 4 and D) is a 90° rotation of domain 4. E) Sequence alignment between Ply (top line) and Pfo (bottom line), centered at the undecapeptide sequence (bold). Areas of homology between the two sequences are shown as <u>underlined</u>. Mutagenesis targets are shown as colored blocks.</p

Ply Oligomerization Behavior.

<p>Ply molecules were incubated either in the presence or absence of HCECs in a total volume of 20 µl before being directly mixed with SDS loading buffer and electrophoresed through a 6% SDS polyacrylamide gel with or without boiling. The gel was blotted to a PVDF membrane, blocked in 5% skim milk, and sequentially labeled with 1° (polyclonal anti-Ply rabbit serum) and 2° (HRP conjugated goat anti-rabbit IgG) antibodies. Ply monomers are shown as 53 kDa bands along with dimers and high molecular weight oligomers.</p

Ply Hemolytic Activity.

<p>Hemolytic activity of Ply was measured in the presence of 2% RBCs. The results are shown as relative % hemolysis. RBCs and 1% TX100 served as the positive control and represents 100% lysis. Each mutant was statistically compared to Ply^WT and significant differences (P<0.05) are denoted as either a single asterisk (open triangles), 2 asterisks (closed triangles), or 3 asterisks (open diamond) to account for each represented mutant. Error bars are not pictured to prevent obstruction of the data points for viewing.</p

Ply Cytotoxicity on HCECs.

<p>Ply cytotoxicity on HCECs was measured by staining with calcein AM, a selective marker for healthy cells with intact membranes. Percent survival was normalized using PBS (negative) and TX100 (positive) treated cells as controls. The results are shown as % survival. Each mutant was statistically compared to Ply^WT and significant differences (P<0.05) are denoted as a single asterisk (open triangles), 2 asterisks (closed triangles), or 3 asterisks (open diamond) to account for each represented mutant. Error bars are not pictured to prevent obstruction of the data points for viewing.</p

Ply Surface Binding to HCECs.

<p>Flow cytometry was used to assess surface binding to HCECs. Various concentrations of AF488 labeled Ply were incubated in the presence of 3x10⁵ fixed HCECs. AF488 labeled bovine serum albumin (BSA) and AF488 labeled heat inactivated (HI) Ply^WT were included as controls. Data is presented as % of cells with Ply surface binding compared to total cells. Each mutant was statistically compared to Ply^WT and significant differences (P<0.05) are denoted as either a single asterisk (open squares) or 2 asterisks (closed circles) to account for each group. Error bars are not pictured to prevent obstruction of the data points for viewing.</p