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Abstract
Streptococcus pneumoniae is widely known for its ability to cause deadly diseases in the brain, lung, and blood of people all over the world. This bacterium is also a common cause of ocular infectious diseases including conjunctivitis, keratitis, and endophthalmitis. Conjunctivitis and keratitis are both ocular surface infections that occur rather frequently, but carry relatively lower risk of vision loss compared to endophthalmitis. Endophthalmitis is an intraocular infection, which occurs less often than surface infection. However, this type of infection carries a higher risk of vision loss. Streptococcal endophthalmitis patients tend to carry a poor prognosis with approximately 30% suffering from significant reductions in visual acuity or enucleation, removal of the entire eye.
Diabetic patients receiving intravitreal injections of anti-inflammatory drugs are among the most common victims of S. pneumoniae endophthalmitis. The intraocular environment of a patient suffering from uncontrolled diabetes contains a high glucose concentration, similar to the blood. We hypothesized that increased glucose concentration in the vitreous humor of diabetic rabbits would lead to greater bacterial recovery than nondiabetic rabbits from the vitreous humor. A population of insulin-dependent type I diabetes (IDID) rabbits as well as nondiabetic rabbits were intraocularly infected with a S. pneumoniae endophthalmitis clinical isolate, E353, for 48 hours. No significant difference in bacteria recovery from the vitreous humor was observed from the two rabbit groups. However, 33% of IDID rabbits developed bacteremia, while 25% of IDID rabbits developed contralateral endophthalmitis (infections in the non-experimentally infected eye). No control rabbits developed bacteremia or contralateral endophthalmitis. This suggested a breakdown in the blood-ocular barrier in the diabetic eye. To better understand how S. pneumoniae is able to proliferate in vitreous at such an expansive rate and cause extensive damage, we used a genome-wide assessment tool known as transposon-sequencing (Tn-seq). A 28,000 member mutant library of the S. pneumoniae strain D39 was grown in nutrient rich media and vitreous humor. Sequencing reads were compared to determine which genes were essential for pneumococcal growth in vitreous humor. One gene deemed essential was an ascorbic acid (AA) transporter subunit (spd_1846) of a phosphotransferase system (PTS). AA is necessary in the host intraocular environment for protection against oxidative damage. It is also one of more than 30 carbon sources S. pneumoniae has been shown to metabolize. The 9 gene operon that contains this essential gene is known as ula, and has been shown to be transcribed as a single unit. It was previously reported that a transcriptional activator controlled the expression of all the genes in the ula operon. Nevertheless, removal of that activator did not attenuate growth of the mutant in AA containing media possibly due to presence of a second AA specific PTS operon, ula2. By removing the transcriptional activator gene, ulaR2, of the second AA specific PTS operon, ula2, we observed significantly reduced bacterial growth of pneumococcal strain D39 in vitreous humor both in vitro and in vivo. D39Δ ulaR2 infected rabbits also had significantly less myeloperoxidase (MPO) activity (an indicator of neutrophil activity) than those infected with D39 at 48 hours post infection. When the same mutation was made in an endophthalmitis clinical isolate, E335, significantly reduced in vitro bacterial growth in vitreous humor was also observed. E335ΔulaR2 showed reduced in vivo bacterial load recovered at 24 hours post infection as well as reduced MPO activity at 48 hours post infection; however, neither data points were statistically significant.
The abundance of glucose, the preferential carbon source, in diabetic vitreous was not the determining factor for expansive growth occurring during endophthalmitis. Tn-seq data suggested six gene candidates that could lead to a better understanding of how the pneumococcus is able to cause damage and proliferate in the intraocular environment. The transcriptional regulator gene, ulaR2, was shown to be essential for optimal growth in vitreous humor both in vitro and in vivo for the pneumococcal strain D39. These studies together have provided insight into the metabolic and genetic factors S. pneumoniae utilize during intraocular growth. A better understanding of the metabolic and genetic factors allowing such massive bacterial proliferation in this specialized environment will increase the comprehensive knowledge of pneumococcal metabolism and pathogenesis for other disease models.
