Nitric oxide effects on GBS gene expression and survival.

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Submission ID :
ESPR293
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Abstract: :

Background: Group B Streptococcus (GBS) remains a major cause of neonatal infectious morbidity and mortality. In some cases, vaginal colonization and subsequent uterine invasion leads to chorioamnionitis prior to delivery. Chorioamnionitis increases the risk of miscarriage, preterm birth, and early-onset sepsis.


GBS possesses virulence factors that allow it to bypass the maternal-fetal defenses of the placenta. One such defense is nitric oxide (NO), an important component of innate immunity, which induces microbial death through several mechanisms. NO is produced by nitric oxide synthase (NOS), and NOS has been found in human trophoblast cells and placental macrophages.


Objectives/hypothesis: This study aims to elucidate the mechanisms that GBS uses to survive NO exposure in vitro and to understand the role of placental NO in host-pathogen interactions in a GBS chorioamnionitis mouse model. We hypothesize that placental NOS activity is increased in chorioamnionitis and that exposure to NO results in GBS up-regulation of pathways to enhance survival in a NO-rich environment.


Methods: To characterize the effect of NO on GBS growth, we measured optical densities of GBS strains from all ten capsular serotypes, seeded and grown to stationary phase in culture with variable concentrations of DETA, a NO donor. Whole-genome GBS transcriptomic profiling was performed on two strains (serotypes Ia and V) under NO exposure or control growth conditions. We also used in vitro hemolysis assays to assess the effect of NO on expression of ß-hemolysin/cytolysin, an important GBS cytolytic virulence factor.


Results: NO exposure resulted in dose-dependent GBS growth inhibition that varied by strain, with one serotype V strain, CNCTC 10/84, showing increased NO resistance compared to the others. RNA-seq showed that strain 10/84 and a serotype Ia comparator shared several pathways that were differentially expressed under NO exposure conditions. Riboflavin biosynthesis showed greater than four-fold expression increases during NO exposure (p<0.001 by differential gene expression analysis). Additionally, strain 10/84 showed significant upregulation of the arginine deiminase pathway (p<0.001 by gene set enrichment analysis), which was not seen in the serotype Ia strain. Increasing NO concentrations led to significantly decreased hemolysis (p<0.0001, one-way ANOVA with Bonferroni correction for multiple comparisons).


Conclusions: NO inhibits GBS growth, but GBS has shared and strain-specific protective mechanisms activated by NO exposure. NO results in decreased GBS ß-hemolysin/cytolysin expression, which may help GBS evade phagocytic destruction. Ongoing work includes mutant library screening to identify bacterial genes that are essential for survival during NO exposure and generation of targeted GBS gene knockdowns and knockouts to validate our transcriptomic results. We are also characterizing placental NOS expression during chorioamnionitis and its role in fetal outcomes.

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UPMC- Children's Hospital of Pittsburgh
UPMC- Children's Hospital of Pittsburgh

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