The Streptococcus pneumoniae capsule is required for full virulence in pneumococcal endophthalmitis

Abstract

Purpose: To determine whether Streptococcus pneumoniae capsule was necessary for pathogenesis of pneumococcal endophthalmitis.

Methods: An isogenic capsule-deficient strain was created using homologous recombination. New Zealand White rabbits were injected intravitreously with 10(2) colony-forming units (CFU) of the parent strain or the capsule mutant. Slit lamp examination (SLE), electroretinography, and myeloperoxidase activity were performed 24 and 48 hours postinfection (PI). Serial dilutions of vitreous were plated to quantitate CFU, eyes were extracted for histology, and host cytokine mRNA expression was determined.

Results: Eyes infected with the parent strain had significantly higher SLE scores than eyes infected with the capsule-deficient strain 24 and 48 hours PI (P < 0.001). CFU recovered from eyes infected with the capsule mutant were significantly fewer than CFU recovered from eyes infected with the parent strain 24 and 48 hours PI (P < 0.001). The parent strain caused a significantly greater decrease in retinal function and more retinal destruction than the mutant strain 48 hours PI (P = 0.026). Vitreal IL-1β, IL-6, and TNF-α were upregulated by both the parent and mutant strain 12 hours PI. By 48 hours PI, there was significantly more neutrophil infiltration in the vitreous infected with the parent strain.

Conclusions: Endophthalmitis caused by the encapsulated strain is more damaging to retinal function and structural integrity. These findings indicate that capsule is an important virulence factor of S. pneumoniae endophthalmitis, in contrast to keratitis, suggesting that the anatomic host site in pneumococcal ocular infections is important.

Figure 1.

(A) Average SLE scores 24 and 48 hours PI. (B) Endophthalmitis caused by the parent strain or capsule-deficient mutant strain at 24 and 48 hours PI. Parent, parent strain; mutant, capsule-deficient isogenic mutant strain. *P < 0.01.

Figure 2.

Percentage loss of retinal function at 24 and 48 hours PI for eyes infected intravitreously with the parent strain or the mutant strain. At 48 hours PI, eyes infected with the parent strain had a significantly higher percentage loss of retinal function than eyes infected with the mutant strain. Error bars represent 95% confidence intervals.

Figure 3.

Cytokine mRNA expression at 3 and 12 hours PI for eyes infected intravitreously with the parent strain (lanes 1–4, 8–11) or the mutant strain (lanes 5–7, 12, 13). IL-1β, TNF-α, and IL-6 were upregulated in the vitreous by both strains. IL-10 was not expressed by 48 hours PI (12 hours PI shown), and IL-8 was expressed at all time points in both infections. GAPDH was the housekeeping gene. Each lane represents vitreous from an individual eye.

Figure 4.

Representative histology sections (hematoxylin and eosin staining) of eyes infected intravitreously with the parent strain (A–E) or the mutant strain (F–J) at 12 (A, F), 24 (B, G), 36 (C, H), and 48 (D, E, I, J) hours PI. At 48 hours PI, the retina of eyes infected with the parent strain (E) showed a far more severe pathology than the retina of eyes infected with the mutant strain (J). Original magnification, 40× (A–D, F–I) and 400× (E, J).

Figure 5.

Representative histology sections (neutrophil staining) of eyes infected intravitreously with the parent strain (A–F) or the mutant strain (G–L) at 12 (A, G), 24 (B, H), 36 (C, I), and 48 (D–F, J–L) hours PI. Neutrophils were observed in vitreous infected with the parent strain at all time points (A–D). Infiltration of neutrophils occurred between 24 and 36 hours PI in vitreous infected with the mutant strain (H, I, respectively). (E, K) Higher magnifications of (D) and (J), respectively, to show morphology. Neutrophils are stained brown. (F, L) Isotype controls. Original magnifications, 20× (A–D, G–J); 1000× (E, F, K, L).

Figure 6.

Representative histology pictures (macrophage staining) of eyes infected intravitreously with the parent strain (A–D) or the mutant strain (E–H) at 48 hours PI. Arrows indicate macrophages (stained brown). (C, G) Magnifications of (B) and (F), respectively, to show morphology. (D, H) Isotype controls. Original magnification, 20× (A, E); 400× (B, F); 1000× (C, D, G, H). Areas from (A) and (E) magnified in (B) and (F) are boxed.