The Streptococcus agalactiae Stringent Response Enhances Virulence and Persistence in Human Blood

Abstract

Streptococcus agalactiae (group B Streptococcus [GBS]) causes serious infections in neonates. We previously reported a transposon sequencing (Tn-seq) system for performing genomewide assessment of gene fitness in GBS. In order to identify molecular mechanisms required for GBS to transition from a mucosal commensal lifestyle to bloodstream invasion, we performed Tn-seq on GBS strain A909 with human whole blood. Our analysis identified 16 genes conditionally essential for GBS survival in blood, of which 75% were members of the capsular polysaccharide (cps) operon. Among the non-cps genes identified as conditionally essential was relA, which encodes an enzyme whose activity is central to the bacterial stringent response—a conserved adaptation to environmental stress. We used blood coincubation studies of targeted knockout strains to confirm the expected growth defects of GBS deficient in capsule or stringent response activation. Unexpectedly, we found that the relA knockout strains demonstrated decreased expression of β-hemolysin/cytolysin, an important cytotoxin implicated in facilitating GBS invasion. Furthermore, chemical activation of the stringent response with serine hydroxamate increased β-hemolysin/cytolysin expression. To establish a mechanism by which the stringent response leads to increased cytotoxicity, we performed transcriptome sequencing (RNA-seq) on two GBS strains grown under stringent response or control conditions. This revealed a conserved decrease in the expression of genes in the arginine deiminase pathway during stringent response activation. Through coincubation with supplemental arginine and the arginine antagonist canavanine, we show that arginine availability is a determinant of GBS cytotoxicity and that the pathway between stringent response activation and increased virulence is arginine dependent.

Cas9 Contributes to Group B Streptococcal Colonization and Disease

“…Upon transmission of GBS to the neonate, however, other GBS factors may contribute to invasive disease progression. During the pathogenesis of meningitis, GBS expresses factors that promote survival in the bloodstream such as capsular polysaccharide (Hooven et al, 2018), the beta-hemolysin/cytolysin (β-h/c) and associated carotenoid pigment (Liu et al, 2004), as well as factors that promote interaction with the blood-brain barrier (BBB), such as surface adhesins BspC (Deng et al, 2019), SfbA (Mu et al, 2014) and pili (Maisey et al, 2007; Banerjee et al, 2011).…”

Regulation of (p)ppGpp and Its Homologs on Environmental Adaptation, Survival, and Pathogenicity of Streptococci

“…Variation of RSH in S. pneumoniae is found to be associated with phenotypic differences based on a genomic diversity analysis, and researchers further confirmed that RSH confers higher resistance to neutrophil-killing (Li et al, 2015). In S. agalactiae, Rel Sag is also essential to its survival in blood (Hooven et al, 2018). These results suggest the important roles of Rel Sag /(p)ppGpp in the resistance to immune killing by the host.…”

“…In S. pneumoniae, Rel Spn and (p)ppGpp amounts play wide-ranging homeostatic roles in pneumococcal physiology, and the operon encoding the major exotoxin pneumolysin is also under the regulation of (p)ppGpp/Rel Spn (Kazmierczak et al, 2009). In S. agalactiae, the transcription levels of the arginine deiminase (arcA) pathway are decreased during stringent response, while arginine availability modulates the expression of cytotoxicity, which is important for virulence (Hooven et al, 2018). During glucose starvation, besides the classic stringent response including inhibition of growth and related bio-macromolecular synthesis, the extended adaptive response includes inhibited glycolysis, and carbon catabolite repression (CCR)-mediated carbohydrate dependent metabolic switches in S. suis in our tests (Zhang et al, 2016).…”

“…In our tests in S. suis , disruption of rel Ss and relQ leads to decreased adhesive and invasive abilities to Hep-2 cells, and mouse infection experiments show that the Δ rel Ss Δ relQ mutant is attenuated and becomes easier to be cleaned up by the host ( Zhu et al, 2016 ). In S. agalactiae , Rel Sag is essential to its survival in blood, and the rel Sag knockout strains demonstrated a decreased expression of beta-hemolysin, which is implicated in invasion of this pathogen ( Hooven et al, 2018 ).…”

Global gene expression analysis ofStreptococcus agalactiaeat exponential growth phase

“…Together with improved computational tools for data analysis, innovations in RNA-seq technologies are contributing to a better understanding of RNA biology and intermolecular interactions that govern RNA function, as well as transcriptional dynamics changes driving bacterial response and adaptation, to distinct growth conditions and external stimuli [9]. Recently, RNA-seq technology has been successfully applied to clarify some molecular mechanisms of pathogenesis in S. agalactiae [8,[10][11][12][13][14][15]. The first S. agalactiae comparative transcriptomic study evidenced several genetic factors (in particular related to lactose metabolism) likely important in adaptation to the bovine environment [10].…”