Site specific mutations of GalR affect galactose metabolism in Streptococcus pneumoniae

Abstract

Streptococcus pneumoniae (the pneumococcus) is a formidable human pathogen that is capable of asymptomatically colonising the nasopharynx. Progression from colonisation to invasive disease involves adaptation to distinct host niches, which vary markedly in the availability of key nutrients such as sugars. We have previously reported that cell-cell signalling via the autoinducer-2 (AI-2)/LuxS quorum sensing system boosts the capacity of S. pneumoniae to utilise galactose as a carbon source by upregulation of the Leloir pathway. This resulted in increased capsular polysaccharide production and a hypervirulent phenotype. We hypothesised that this effect was mediated by phosphorylation of GalR, the transcriptional activator of the Leloir pathway. GalR is known to possess three putative phosphorylation sites: S317, T319 and T323. In the present study, derivatives of S. pneumoniae D39 with putative phosphorylation-blocking alanine substitution mutations at each of these GalR sites (singly or in combination) were constructed. Growth assays and transcriptional analyses revealed complex phenotypes for these GalR mutants, with impacts on the regulation of both the Leloir and tagatose 6-phosphate pathways. The alanine substitution mutations significantly reduced the capacity of pneumococci to colonize the nasopharynx, middle ear and lungs in a murine intranasal challenge model. Importance: Pneumococcal survival in the host and capacity to transition from a commensal to a pathogenic lifestyle is closely linked to its ability to utilise specific nutrients in distinct niches. Galactose is a major carbon source for pneumococci in the upper respiratory tract. We have shown that both the Leloir and tagatose-6-phosphate pathways are necessary for pneumococcal growth in galactose, and demonstrate GalR-mediated interplay between the two pathways. Moreover, the three putative phosphorylation sites in the transcriptional regulator GalR play a critical role in galactose metabolism and are important for pneumococcal colonization of the nasopharynx, middle ear and lungs.