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

Abstract

International audienceNasopharyngeal colonisation by Streptococcus pneumoniae is characterised by adherence to epithelial cells, microinvasion and innate immune activation. Previously, we have shown that two biosynthesis gene mutants of S. pneumoniae serotype 6B strain ( ΔproABC and Δfhs with an additional ΔpiaA mutation) can successfully colonise murine and human models without causing disease. Here, we test the hypothesis that epithelial microinvasion and an innate immune response persist despite disease attenuation. We show that although the ΔproABC and Δfhs mutations do not attenuate microinvasion in either experimental human pneumococcal challenge or infection of epithelial cell models, there was less transmigration of Detroit 562 nasopharyngeal epithelial cells by the biosynthesis gene mutants than the WT. Cellular reorganisation by primary human airway epithelial cells varied considerably between strains. Compared to the WT, infection of Detroit 562 epithelial cells by the Δfhs/piaA mutant but not the ΔproABC/piaA mutant was less pro-inflammatory, induced less caspase 8 production, associated increased pneumococcal hydrogen peroxide secretion and reduced pneumolysin activity. Under serum stress, these biosynthesis gene mutations had a broad impact on the expression of pneumococcal virulence genes (e.g. ply , nanA and psaA ), those regulating oxidative stress (e.g. SpxB , lctO and adhE ), and genes involved in purine and carbohydrate metabolism. However, although these may result in disease attenuation, they were not directly linked to effects on microinvasion, cellular reorganisation or the epithelial innate immune response. These findings suggest that the strain differences observed were driven by the differential expression of multiple bacterial virulence and metabolic pathways, rather than any single gene or pathway 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. Author Summary Streptococcus pneumoniae (the pneumococcus) commonly colonises the back of the human nose, and is a leading cause of pneumonia, meningitis, and sepsis. During colonisation, the pneumococcus adheres to the cells in the nose, invades these cells (so-called microinvasion), and activates them. Colonisation is a pre-requisite for disease, however, since disease is largely a dead end for S. pneumoniae , it remains unclear whether these processes are directly linked to disease progression. We have previously shown that if we introduce gene mutations into S. pneumoniae that affect key metabolic pathways, these bacteria retain their ability to colonize human and animal models without causing disease. We now show that these mutants retain their ability to microinvade epithelial cells, and some may still cause inflammation, but are less able to pass through the epithelial barrier. However, although the attenuation of disease may be explained by the broad-ranging impact of these mutations on pneumococcal virulence, oxidative stress, and metabolism, they are not driven by a single determinant. Our findings suggest that pneumococcal microinvasion and immune activation are not necessarily pre-cursors to disease progression. This supports the idea that S. pneumoniae adapts and evolves to promote colonisation and ultimately transmission rather than cause disease. Graphical Abstract S. pneumoniae colonisation is characterised by mucus association, epithelial adherence, microcolony formation and microinvasion – where the pneumococcus invades the epithelial barrier without causing disease. Although mutations in S. pneumoniae biosynthesis genes ( ΔproABC and Δfhs ) attenuate disease in a murine model, they do not attenuate microinvasion in either experimental human pneumococcal challenge (EHPC) or in vitro in primary and immortalised epithelial cells. Transmigration of the epithelial barrier is attenuated. These mutations show strain-dependent effects on both the epithelial and bacterial responses to infection. Factors such as epithelial cellular reorganisation, inflammation and caspase 8 activity alongside pneumococcal metabolic adaptation, virulence factor expression and response to stress are important components of these processes