Effect of peptidoglycan modifying enzymes PgdA and Adr on relative fitness during murine colonization in the presence and absence of lysozyme M.

<p>LysM^+/+ and LysM^−/− mice were challenged with equal inocula of the wild-type (WT) strain or revertant and the defined mutant indicated, and the density of each strain was determined in upper respiratory tract lavages 3 days post-inoculation. Each symbol represents the competitive index value for an individual animal. The competitive index was calculated based on the ratio of mutant to WT bacteria in nasal lavages compared to the ratio of mutant to WT bacteria in the inoculum. The dotted line is at a value of one; a value greater than one indicates the mutant out-competes the WT, a value less than or equal to one indicates the WT out-competes the mutant. A) <i>pgdAadr</i> vs. WT (^*** p = 0.001). B) <i>pgdAadr</i> vs. the revertant (<i>pgdA+ adr+</i>) (^** p = 0.004). C) <i>pgdA</i> vs. WT. D) <i>adr</i> vs. WT.</p

Effect of peptidoglycan modifying enzymes PgdA and Adr on hydrolysis of pneumococcal cell walls in the presence of lysozyme.

<p>Hydrolysis of peptidoglycan (50 µg/ml), purified from the wild-type (WT) strain or the defined mutants indicated with lysozyme (100 µg/ml) from A) chicken egg (+L) or B) human (+Hu L). Representative experiment showing percentage of hydrolysis based on the optical density (OD 600 nm) of each reaction at time 0 min.</p

Effect of peptidoglycan modifying enzymes PgdA and Adr on growth of pneumococci in the presence or absence of lysozyme.

<p>Growth characteristics of the wild-type (WT) strain or defined mutants were compared by following the optical density (OD 620 nm). Once the broth culture reached mid-log phase, lysozyme (100 µg/ml) was added where indicated by an arrow. A) chicken egg lysozyme (+L) or B) recombinant human lysozyme (+Hu L). Graphs are representative of six independent experiments.</p

Sensitivity to recombinant human lysozyme.

<p>Sensitivity is shown by the estimated MBC₅₀ (mean bactericidal concentration) of the wild-type (WT) strain and defined mutants. Strains tested were in a <i>lytA</i> background to eliminate effects of autolysis. Ranges were based on three independent determinations.</p

Expression of lysozyme M in the mouse nasopharyx.

<p>A) Western blot of nasal lavages from mice colonized with the wild-type strain for two days incubated with antisera to lysozyme M. Lane 1: LysM^+/+, rat IgG control. Lane 2: LysM^+/+, neutrophil depletion with mAb RB6-8C5. Lane 3: LysM^−/−. Size markers are in kilodaltons. B) Immunohistochemistry on paraffin-embedded tissue sections through the nasal tissue 24 hrs post-inoculation with the wild-type strain. Staining with i) anti-lysozyme M (LysM^+/+), ii) no primary antibody control (LysM^+/+), and iii) anti-lysozyme M (LysM^−/−). Sections were counterstained with hematoxylin. Magnification 400×.</p

Effect of peptidoglycan modifying enzymes PgdA and Adr on viability of pneumococci in the presence of lysozyme.

<p>Once the broth culture of the wild-type (WT) strain or the defined mutants indicated reached mid-log phase, lysozyme (100 µg/ml) was added and viable counts (CFU/ml) were measured 5 hrs later. Strains tested were in a <i>lytA</i> background to eliminate effects of autolysis. Conditions included A) chicken egg lysozyme (+L) and B) recombinant human lysozyme (+Hu L) or heat inactivated recombinant human lysozyme (+Hu IL). Graphs are based on four independent determinations ±S.D. (^* p<0.05).</p

The contribution of lysozyme from neutrophils to survival and colonization of mutants lacking peptidoglycan modifications.

<p>A) Neutrophils isolated from human blood were incubated with serum opsonized bacteria and survival was assessed following a 45 min incubation. Percent survival was calculated based on viable counts (CFU/ml) relative to no neutrophil controls. (^*** p<0.001). B) Neutrophils were depleted with anti-Ly6G antibody RB6-8C5 prior to challenge with an equal inoculum of <i>pgdAadr</i> and wild-type (WT) strains. Controls received rat IgG. Two days post-inoculation nasal lavages were obtained to quantify the competitive index. (^** p<0.01).</p

Effect of peptidoglycan modifying enzymes PgdA and Adr on expression of capsular polysaccharide (CPS).

<p>Sonicates of the bacterial strain indicated were used in a capture ELISA to measure the amount of cell-associated type 4 CPS produced relative to the total amount of protein. Values are relative to a standard with purified type 4 CPS and based on four independent determinations ±S.D. (^* p<0.05, ^** p<0.01).</p

Predicted peptidoglycan structure.

<p>PgdA, an <i>N</i>-acetyl glucosamine, and Adr, an <i>O</i>-acetyltransferase, modify the MurNAc-GlcNAc disaccharide structure at the indicated sites. The major pneumococcal autolysin, LytA, cleaves the stem peptide attached to MurNAc. Lysozyme hydrolyzes the glycosidic bond between MurNAc and GlcNAc as shown.</p

FQE increases CpsD-P in <i>S. pneumoniae</i> D39.

<p><i>S. pneumoniae</i> D39 were grown to mid log phase in THY (OD₆₀₀ ≈ 0.35) and FQE at indicated concentrations were added. (A) These concentrations (µM) had no statistically significant effect on CFU/ml after 30, 60 and 120 mins. (B) Whole cell lysates were prepared from these cells, which were separated by SDS-PAGE and analyzed by immunoblotting using anti-CpsD, or anti-phosphotyrosine (to detect CpsD-P). (C) Densitometric analysis of CpsD-P from three separate experiments. The effect with addition of 5 µM was significantly higher than compared with 1.25 µM FQE (* - <i>P</i><0.05 by Student’s <i>t</i>-test). (D) For comparison, the effect of an in-frame <i>cpsB</i> deletion mutant on CpsD-P is shown.</p