Disease-attenuated pneumococcal biosynthesis gene mutants invade the mucosal epithelium and induce innate immunity

Weight, Caroline M. and Pollara, Gabriele and Betts, Modupeh and Ragazzini, Roberta and Ramos-Sevillano, Elisa and Reiné, Jesús and Whelan, Matthew and Guerra-Assunção, José Afonso and Connor, Michael and Bonfanti, Paola and Jolly, Clare and Noursadeghi, Mahdad and Ferreira, Daniela M. and Brown, Jeremy S. and Heyderman, Robert S. (2023) Disease-attenuated pneumococcal biosynthesis gene mutants invade the mucosal epithelium and induce innate immunity. Other. bioRxiv.

Abstract

Nasopharyngeal colonisation by Streptococcus pneumoniae is characterised by bacterial adherence to epithelial cells, microinvasion and innate immune activation. Previously, we have shown two serotype 6B S. pneumoniae mutant strains affecting bacterial metabolism (ΔproABC/pia and Δfhs/pia) colonise humans and mice, but in a murine disease model do not cause invasive infection. Here, we explore whether S. pneumoniae epithelial microinvasion and the induction of innate immune responses persist despite disease attenuation. We show that under serum stress, these biosynthesis gene mutations had a broad but different impact on pneumococcal virulence gene expression, oxidative stress regulation, and purine and carbohydrate metabolism genes. However, although these mutations did not attenuate microinvasion in human challenge and epithelial models, there was less transmigration of Detroit 562 nasopharyngeal epithelial cells by the mutants compared to WT. Cellular reorganisation of primary human airway epithelium varied considerably between strains. Compared to WT, infection of Detroit 562 epithelial cells by the Δfhs/piaA strain, but not the ΔproABC/piaA strain was less pro-inflammatory, induced less caspase 8 production, and were associated with increased pneumococcal hydrogen peroxide and reduced pneumolysin secretion. These findings suggest that the observed differences in microinvasion and the epithelial response were driven by the differential expression of multiple bacterial virulence and metabolic pathways, rather than single genes or pathways of genes. These data highlight the complex impact of single gene mutations on bacterial virulence and suggest that the virulence determinants of pneumococcal epithelial colonisation, microinvasion and innate immunity are not necessarily directly linked to disease.