The proteomic and transcriptomic landscapes altered by Rgg2/3 activity in Streptococcus pyogenes

Abstract

Streptococcus pyogenes, otherwise known as Group A Streptococcus (GAS), is an important and highly adaptable human pathogen with the ability to cause both superficial and severe diseases. Understanding how S. pyogenes senses and responds to its environment will likely aid in determining how it causes a breadth of diseases. One regulatory network involved in GAS’s ability to sense and respond to the changing environment is the Rgg2/3 quorum sensing (QS) system, which responds to metal and carbohydrate availability and regulates changes to the bacterial surface. To better understand the impact of Rgg2/3 QS on S. pyogenes physiology, we performed RNA-seq and TMT-LC-MS/MS analysis on cells in which this system was induced or disrupted. Primary findings confirmed that pheromone stimulation in wildtype cultures is limited to the induction of operons whose promoters contain previously determined Rgg2/3 binding sequences. However, supplementing exogenous pheromone to a deletion mutant of rgg3, a strain that endogenously produces elevated amounts of pheromone, led to extended alterations of the transcriptome and proteome, ostensibly by stress-induced pathways. Under such exaggerated pheromone conditions (Δrgg3+SHP), a connection was identified between Rgg2/3 and the stringent response. Mutation of relA, the bifunctional guanosine tetra- and penta-phosphate nucleoside synthetase/hydrolase, and alarmone synthase genes sasA and sasB, impacted culture doubling times and disabled induction of Rgg2/3 in response to mannose, while manipulation of Rgg2/3 signaling modestly altered nucleotide levels. Our findings indicate that excessive pheromone production or exposure places stress on GAS resulting in an indirect altered proteome and transcriptome beyond primary pheromone signaling.IMPORTANCEStreptococcus pyogenes causes several important human diseases. This study evaluates how the induction or disruption of a cell-cell communication system alters S. pyogenes’s gene expression and, in extreme conditions, its physiology. Using transcriptomic and proteomic approaches, the results define the pheromone-dependent regulon of the Rgg2/3 quorum sensing system. In addition, we find that excessive pheromone stimulation, generated by genetic disruption of the system, leads to stress responses that are associated with the stringent response. Disruption of this stress response affects the ability of the cell-cell communication system to respond under certain conditions. These findings assist in the determination of how S. pyogenes is impacted by and responds to non-traditional sources of stress.

Quorum sensing regulation of a major facilitator superfamily transporter affects multiple streptococcal virulence factors

“…Our current study identified a QS-regulated transporter, Spy49_0460, whose expression leads to decreased transcription of at least three secreted virulence proteins: the pore-forming cytolysin, streptolysin O; the chemokine-cleaving protease, SpyCEP; and the antiphagocytic surface antigen, M protein. In support of these observations, a recent proteomics study from our lab confirmed that streptolysin O and M49 are significantly less abundant in the secreted fraction of proteins from QS-induced cells 18 . Spy49_0460 was previously identified in whole genome screens for genes important for virulence in a zebrafish model of necrotizing fasciitis 19, 20 , survival in human blood 21 and pharyngitis in a nonhuman primate model 9 , but the mechanisms underlying its importance in these models have not been determined.…”

“…This suggests that the downregulation of slo, spyCEP, and emm49 transcription could be due to an accumulation of changes in cell physiology or stress that lead to the observed phenotype. In support of this idea, our recent proteomics study also detected nucleotide stress and a link to the stringent response in QS-ON cells 18 . Alternatively, the Rgg2/3 regulon is induced within one hour of infection of the murine nasopharynx 10 , though little is known about its expression later in infection.…”