Abstract
Purpose :
Streptococcus pneumoniae is a leading cause of endophthalmitis and grows robustly in rabbit vitreous humor. Factors involved in S. pneumoniae fitness in the eye are unknown. The purpose of this study was to identify and target bacterial genes required for ocular growth.
Methods :
An S. pneumoniae transposon insertion library was inoculated into rich bacteriological medium, grown to logarithmic phase, then grown in rabbit vitreous humor in vitro (n=3). Bacterial genomic DNA harvested from both rich medium and vitreous was subjected to transposon-sequencing (Tn-Seq) analysis. Sequencing reads were compared between both populations to determine genes essential for fitness in vitreous. An isogenic mutant deficient in a transcription factor responsible for activation of one of the essential genes was created. Growth of wild type and deletion strains was examined in nutrient-rich media, minimal media (MM), and vitreous humor (n=3). New Zealand white rabbits were infected intravitreally with 102 CFU wild type (n = 8) or isogenic mutant (n =8) strain. Biomicroscopy scores, bacterial burden, and myeloperoxidase activity were determined 24 and 48 hours after infection.
Results :
Tn-Seq identified 6 genes essential for growth in vitreous, one of which encodes an ascorbic acid (AscA) transporter subunit of a phosphotransferase system. An isogenic deletion mutant of spd_1961, encoding the transcription activator of the phosphotransferase system operon, was constructed. There were no significant differences in growth between strains in nutrient-rich media, MM, or MM with supplemental AscA in vitro. However, in vitro growth of the knockout strain was significantly decreased in the vitreous humor at 6, 8, 12, and hours compared to the parent strain (p≤0.025). The growth of the knockout strain was also significantly decreased in vivo at 24 and 48 hours (p≤0.010). Biomicroscopy scores and myeloperoxidase activity were not significantly different between groups. However, eyes infected with wild type bacteria had a significant increase (p=0.043) in myeloperoxidase activity between 24 hours and 48 hours, while eyes infected with the mutant strain did not.
Conclusions :
S. pneumoniae deficient in transcriptional activation of AscA transport has significantly reduced growth in vitreous humor, indicating that AscA is an important carbon source for this bacterium in the intraocular environment.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.