The Role of luxS in the Middle Ear Streptococcus pneumoniae Isolate 947

The LuxS protein, encoded by luxS, is required for the production of autoinducer 2 (AI-2) in Streptococcus pneumoniae. The AI-2 molecule serves as a quorum sensing signal, and thus regulates cellular processes such as carbohydrate utilisation and biofilm formation, as well as impacting virulence. The role of luxS in S. pneumoniae biology and lifestyle has been predominantly assessed in the laboratory strain D39. However, as biofilm formation, which is regulated by luxS, is critical for the ability of S. pneumoniae to cause otitis media, we investigated the role of luxS in a middle ear isolate, strain 947. Our results identified luxS to have a role in prevention of S. pneumoniae transition from colonisation of the nasopharynx to the ear, and in facilitating adherence to host epithelial cells.Alexandra Tikhomirova, Erin B. Brazel, Kimberley T. McLean, Hannah N. Agnew, James C. Paton and Claudia Trappett

Streptococcus pneumoniae Strains Isolated From a Single Pediatric Patient Display Distinct Phenotypes

Streptococcus pneumoniae is the leading cause of bacterial paediatric meningitis after the neonatal period worldwide, but the bacterial factors and pathophysiology that drive pneumococcal meningitis are not fully understood. In this work, we have identified differences in raffinose utilization by S. pneumoniae isolates of identical serotype and sequence type from the blood and cerebrospinal fluid (CSF) of a single pediatric patient with meningitis. The blood isolate displayed defective raffinose metabolism, reduced transcription of the raffinose utilization pathway genes, and an inability to grow in vitro when raffinose was the sole carbon source. The fitness of these strains was then assessed using a murine intranasal infection model. Compared with the CSF isolate, mice infected with the blood isolate displayed higher bacterial numbers in the nose, but this strain was unable to invade the ears of infectedmice. A premature stop codon was identified in the aga gene in the raffinose locus, suggesting that this protein likely displays impaired alpha-galactosidase activity. These closely related strains were assessed by Illumina sequencing, which did not identify any single nucleotide polymorphisms (SNPs) between the two strains. However, these wider genomic analyses identified the presence of an alternative alpha-galactosidase gene that appeared to display altered sequence coverage between the strains, which may account for the observed differences in raffinose metabolic capacity. Together, these studies support previous findings that raffinose utilization capacity contributes to disease progression, and provide insight into a possible alternative means by which perturbation of this pathway may influence the behavior of pneumococci in the host environment, particularly in meningitis.Hannah N. Agnew, Erin B. Brazel, Alexandra Tikhomirova, Mark van der Linden, Kimberley T. McLean, James C. Paton, and Claudia Trappett

Uncovering the link between the SpnIII restriction modification system and LuxS in Streptococcus pneumoniae meningitis isolates

Streptococcus pneumoniae is capable of randomly switching their genomic DNA methylation pattern between six distinct bacterial subpopulations (A-F) via recombination of a type 1 restriction-modification locus, spnIII. These pneumococcal subpopulations exhibit phenotypic changes which favor carriage or invasive disease. In particular, the spnIIIB allele has been associated with increased nasopharyngeal carriage and the downregulation of the luxS gene. The LuxS/AI-2 QS system represent a universal language for bacteria and has been linked to virulence and biofilm formation in S. pneumoniae. In this work, we have explored the link between spnIII alleles, the luxS gene and virulence in two clinical pneumococcal isolates from the blood and cerebrospinal fluid (CSF) of one pediatric meningitis patient. The blood and CSF strains showed different virulence profiles in mice. Analysis of the spnIII system of these strains recovered from the murine nasopharynx showed that the system switched to different alleles commensurate with the initial source of the isolate. Of note, the blood strain showed high expression of spnIIIB allele, previously linked with less LuxS protein production. Importantly, strains with deleted luxS displayed different phenotypic profiles compared to the wildtype, but similar to the strains recovered from the nasopharynx of infected mice. This study used clinically relevant S. pneumoniae strains to demonstrate that the regulatory network between luxS and the type 1 restriction-modification system play a key role in infections and may support different adaptation to specific host niches.Hannah N. Agnew, John M. Atack, Ann R.D. Fernando, Sophie N. Waters, Mark van der Linden, Erin Smith, Andrew D. Abell, Erin B. Brazel, James C. Paton, and Claudia Trappett