Exploring Genomic, Phenotypic, and Epigenetic Characteristics of Clinical Isolates of Streptococcus pneumoniae

Abstract

Streptococcus pneumoniae (the pneumococcus) is a prevalent nasopharyngeal commensal in humans that can transition from a harmless coloniser to a dangerous pathogen, contributing significantly to global bacterial infections and associated morbidity and mortality. Unfortunately, the intricate mechanisms influencing individual strain capabilities to cause localized or invasive pneumococcal diseases remain enigmatic, primarily due to the extensive genetic diversity between pneumococcal strains, with over 100 capsular serotypes superimposed on more than 12,000 clonal sequence types (STs). This study builds upon prior research that used closely related strains, of the same serotype and ST, isolated from the blood and ears of patients to investigate factors influencing disease progression and outcome. Specifically, it was found in serotype 14 ST15 and serotype 3 ST180 blood and ear isolates pairs, that the ability to metabolise the carbohydrate raffinose, influenced by a single nucleotide polymorphism (SNP), resulted in distinct disease progression within an intranasal murine infection model (Minhas et al., 2019). This emphasises the importance of investigating minor disparities between closely related clinical isolates, whilst bypassing the significant genetic diversity inherent in S. pneumoniae. In this project, a collection of clinical isolates was screened for carbohydrate metabolism. Among these, a pair of closely related pneumococcal isolates (serotype 15C, ST 8711) from the blood and cerebrospinal fluid (CSF) of a single paediatric meningitis patient exhibited significant differences in raffinose metabolism. The blood-derived isolate (60B) displayed defective raffinose metabolism, reduced gene transcription in the raffinose utilisation pathway and inability to use raffinose as a carbon source. In a murine intranasal infection model, 60B displayed higher bacterial counts in nasal tissue, compared to those infected with 60CSF, but couldn't survive in the ears of infected mice. Genome sequencing revealed a premature stop codon in the aga gene within the raffinose locus of both strains, suggesting the presence of impaired alpha-galactosidase activity in both strains. Moreover, genome analyses revealed no SNPs between these closely related strains, implying other factors were involved. As genome sequencing did not provide a molecular basis for the observed differences, the spnIII type I restriction-modification system, known for its role in methylation and subsequent gene regulation, was explored. Recombination of the spnIII locus leads to six different variants (A-F) of a methyltransferase, each with a distinct methylation pattern, resulting in unique bacterial subpopulations with varying phenotypic characteristics. Notably, spnIIIB has been associated with increased nasopharyngeal carriage and downregulation of the LuxS/AI-2 Quorum Sensing (QS) system (Manso et al., 2014), a universal communication channel among bacteria linked to virulence and biofilm formation in pneumococci (Trappetti et al., 2017; Trappetti, Potter, et al., 2011; Yadav et al., 2018). The analysis of spnIII alleles in both strains showed a higher proportion of spnIIIB in 60B, correlating with reduced luxS expression. Deletion of luxS in both strains showed altered metabolic profiles and in vivo behaviour. Furthermore, this study revealed, for the first time, that spnIII not only influences LuxS production but also that LuxS, through an unknown mechanism, affects the proportion of spnIII alleles, potentially influencing disease progression in mice. Collectively, these discoveries indicate that closely related Streptococcus pneumoniae isolates from a paediatric meningitis patient's blood and CSF demonstrate divergent raffinose metabolism, which correlates with disease outcomes in a murine model. Furthermore, it highlights the importance of the regulatory interplay between luxS and the spnIII type I restriction-modification system in shaping disease progression and outcomes.