Bacterial adaptive responses to environmental fluctuations, such as decreased nutrient availability, can trigger changes in virulence. Streptococcus agalactiae (GBS) is a common commensal organism of the gastrointestinal and vaginal tracts, where it rarely causes disease in healthy hosts. In some cases, however, vaginal colonization during pregnancy can lead to chorioamnionitis, preterm birth, and neonatal sepsis. We hypothesize that perinatal changes in micronutrient availability lead to upregulation of the pigmented GBS toxin beta-hemolysin/cytolysin (βHC), which we have shown to be important in the pathogenesis of ascending chorioamnionitis.

We performed coincubation screens on amino acids and derivatives that might serve as structural precursors to βHC, as well as compounds that could induce bacterial stress responses through metabolic derangement. We performed shotgun genome sequencing and de novo assembly of GBS strain 10/84, which is “hypervirulent,” with constitutive upregulation of βHC. We compared the 10/84 genome to that of strain A909, which is not hypervirulent, seeking polymorphisms that might explain the phenotype. Polymorphisms of interest were cloned into the shuttle vector pDCerm, and transformed into 10/84 and A909 using electroporation. Knockout strains were generated through homologous recombination with gene fragments cloned into the temperature-sensitive vector pHY304. Finally, whole transcriptome analysis of 10/84 and A909 was performed using RNA-seq on samples collected from bacteria under stress and non-stress conditions.

Supplementing 10/84 but not A909 with 10 mM L-arginine (L-Arg) increased pigmentation and cytotoxicity by 30% (p<0.05, t test). The effect was suppressible with 10 mM canavanine, an inhibitor of arginine deiminase, as well as supplemental glucose, and was not seen following supplementation with D-Arg or other Arg analogs. Methotrexate, serine hydroxamate, and mupirocin also induced hyperpigmentation in both 10/84 and A909. These compounds all induce a conserved bacterial stress reaction called the stringent response (SR), which is known to upregulate Arg uptake/biosynthesis and prevent its catabolism. Comparative genomics revealed in 10/84 a unique 10-base pair deletion in the promoter region of a two-gene monocistronic operon encoding the Arg metabolic enzymes ArgF and ArcC. 10/84 trans-complemented with a second copy of its own promoter/argF sequence had 10-fold increased βHC production and cytotoxicity (p<0.0001, t test). Knockout strains deficient in the SR genes relA and codY exhibited decreased βHC production and 3- to 6-fold less hemolysis (p<.0001 by t test for both). Transcriptome analysis showed significant SR effects on expression of genes involved in Arg metabolism.

Our results support a GBS adaptive response to micronutrient fluctuation that triggers the SR, increasing intracellular Arg, leading to increased toxin expression. This pathway may play a role in pathogenesis of perinatal infection, and may suggest new treatment/prevention approaches.