Systemic pneumococcal infections are a major cause of morbidity and mortality. Pneumococci isolated from bacteraemia lack clear genetic signatures distinguishing them from isolates recovered from other disease sites. The bloodstream is an evolutionary dead end, providing no means of onward transmission, and acute bacteraemic events offer limited opportunity for within-host evolution. Nonetheless, we reasoned that it might be possible to identify genetic determinants of virulence in blood through genomic comparison of matched lung and blood isolates from individual infections. Using samples from a mouse invasive pneumonia model, we observed frequent occurrence of low population frequency mutations in ilvD - encoding a branched-chain amino acid (BCAA) biosynthesis protein - in bloodstream isolates. These mutations were absent from the lung-resident bacterial population. The single-nucleotide polymorphisms in ilvD clustered in a short stretch of nucleotides that are highly conserved across bacteria, suggesting they might disrupt protein function. Deletion of ilvD or introduction of identified SNPs in pneumococcal strain D39 conferred competitive advantages in both in vitro environments mimicking the bloodstream and in mouse bacteraemia models. Improved survival of ilvD mutants within phagocytic cells was dependent on iron availability. Conversely, disruption of ilvD reduced fitness in the nasopharynx, suggesting BCAA biosynthesis is required for upper airway colonisation but is a liability in the context of systemic infection. Nasopharyngeal colonisation defects could be rescued by administering intranasal BCAA to infected mice, suggesting loss of capacity for de novo BCAA synthesis in ilvD mutants accounted for reduced upper airway fitness. In support of a critical role for BCAA biosynthesis in the primary commensal lifestyle of pneumococcus, genomic analysis of clinical isolates demonstrated that ilvD is under purifying selection. Collectively, these findings highlight the context-specific role of IlvD and BCAA biosynthesis in infection, with a requirement for protein function in the BCAA-restricted environment of nasopharynx, but fitness benefits deriving from loss of IlvD and its associated iron-sulfur cluster, in the BCAA-rich conditions of blood.
Kumar, S., Green, A. E., Lorenz, L. J., Newbold, K., Ball, G., Khlebutina, E., Lo, S., Neill, D. R.
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