Lynskey, Nicola

Gally, David

Rand, Joshua

2025-10-01T10:12:28Z

2025-10-01T10:12:28Z

2025-10-01

Streptococcus pyogenes (S. pyogenes) is a Gram-positive human pathobiont responsible for a diverse spectrum of infections, including scarlet fever, necrotizing fasciitis, and rheumatic fever. A new clonal lineage of emm4 S. pyogenes—designated Clade B—has been reported in Europe and the United States, distinct from the previously dominant Clade A. Clade B is associated with enhanced virulence, yet the molecular mechanisms underlying its evolutionary success remain largely unexplored. This thesis investigates key genetic and phenotypic adaptations in Clade B, particularly the degradation of prophage elements and the emergence of a novel fusion protein, emm::enn, and their implications for bacterial fitness. Through comparative in silico and phenotypic analyses of Clade A and Clade B emm4 isolates, we identified a defining genomic feature of Clade B: a rearrangement of genome segments around ribosomal RNA operons, leading to altered genome architecture and a shifted positioning of the terminus. The prophage elements present within Clade B strains are degraded rendering them cryptic, however the impact of this on streptococcal fitness has not been robustly assessed. To address this, prophage induction assays using mitomycin C were performed and showed that while intact prophages in Clade A isolates were excisable, the degraded prophages in Clade B remained unresponsive. This loss of inducibility significantly reduced prophage-mediated bacterial lysis, suggesting a survival advantage for the emergent lineage. RNA sequencing further revealed that genes within the spd3-carrying prophage were highly expressed upon induction in Clade A but remained transcriptionally silent in Clade B. To elucidate the molecular mechanisms that underpin prophage induction in S. pyogenes, we generated isogenic mutants in a representative Clade A strain, targeting CovS a key regulatory protein and RecA, a well-characterized regulator of DNA damage-induced prophage activation in Gram-negative bacteria. Interestingly, deletion of covS suppressed spd3 prophage expression, enhancing bacterial survival upon mitomycin C exposure. In contrast to previous studies, recA deletion did not inhibit prophage induction; rather, transcriptomic analysis revealed upregulation of prophage-associated genes, suggesting a previously unrecognized RecA-independent pathway of DNA damage-mediated prophage induction. Furthermore, we investigated biofilm formation as a potential contributor to Clade B fitness. Biofilm quantification assays demonstrated a significant increase in biofilm formation in Clade B compared to Clade A, implicating the novel emm::enn fusion protein in this phenotype. Heterologous expression of emm::enn in Lactococcus lactis confirmed its role in promoting biofilm formation, while proteinase K treatment suggested a protein-mediated mechanism. Adhesion assays using tonsil keratinocytes revealed that emm and enn contributed to S. pyogenes adherence to the host epithelium by Clade A but not Clade B isolates, suggesting functional divergence between the lineages, possibly mediated by the novel emm::enn fusion protein. Taken together, our findings reveal that Clade B emm4 S. pyogenes have undergone genetic adaptations that enhance bacterial fitness. The degradation of prophages reduces prophagemediated lysis enhancing in vivo survival, while the acquisition of novel genetic elements, such as emm::enn, promotes biofilm formation. Moreover, this study identifies CovS as a previously unrecognized regulator of prophage induction, and challenges the prevailing notion that S. pyogenes prophage activation is entirely RecA-dependent. These findings highlight an additional selection pressure acting on CovS in vivo and accentuates an alarming trajectory toward increased persistence and virulence, with potential implications for S. pyogenes epidemiology and treatment strategies.

https://hdl.handle.net/1842/44010

http://dx.doi.org/10.7488/era/6538

en

The University of Edinburgh

J., RAND, J. D., JOHNSTON, A. B. E., BOWEN, C. & LYNSKEY, N. N. 2023. Methylome-dependent transformation of emm1 group A streptococci. mBio, 14, e0079823.

2026-10-01

Streptococcus pyogenes

S. pyogenes

Clade B

prophages

emm::enn

prophage activation

bacteria regulation

CovS

RecA

Contribution of prophage degradation and a novel fusion protein to the success of emm4 Streptococcus pyogenes

The contribution of prophage degredation and a novel fusion protein to the success of emm4 Streptococcus pyogenes

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy

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