Epithelial microinvasion and immunity elicited by disease-attenuated pneumococcal biosynthesis gene mutants
Authors: Weight Caroline M. , Pollara Gabriele , Betts Modupeh , Ragazzini Roberta , Sevillano Elisa Ramos- , Reiné Jesús , Whelan Matthew , Guerra-Assunção José Afonso , Connor Michael , Bonfanti Paola , Jolly Clare , Noursadeghi Mahdad , Ferreira Daniela M. , Brown Jeremy S. , Heyderman Robert S. 
Published: June 2023

ABSTRACTNasopharyngeal colonisation byStreptococcus pneumoniaeis characterised by adherence to epithelial cells, microinvasion and an innate immune response. Here, we explore the hypothesis that pneumococcal virulence has primarily evolved to promote these colonisation processes and subsequent transmission, rather than to cause disease. We have used aS. pneumoniaeserotype 6B strain, alongside two biosynthesis gene mutants (ΔproABCandΔfhswith an additionalΔpiaAmutation) known to colonise murine and human models without causing disease. We show that these mutations do not attenuate microinvasion in either experimental human pneumococcal challenge orin vitroin primary and immortalised epithelial cells. However, interactions and subsequent cellular reorganisation by primary human airway epithelial and Detroit 562 nasopharyngeal cells were strain dependent, and the inflammatory epithelial innate response and caspase 8 activity were attenuated by theΔfhs/piaAbut not theΔproABC/piaAmutation. Growth of theΔfhs/piaAmutant was associated with increased pneumococcal hydrogen peroxide secretion and reduced pneumolysin activity when compared to the WT andΔproABC/piaAmutant strains. Analysis of the bacterial transcriptome under stress conditions revealed that the mutations resulted in broad, differential effects on pneumococcal virulence and metabolic gene expression suggestive of compensatory changes that preserve microinvasion. The findings that cellular reorganisation, the innate immune response, and the ability to cause invasive disease are not necessarily interconnected emphasise the complexity of bacterial-host interactions at the mucosal surface. Colonisation outcome cannot be fully explained by single or functionally grouped virulence or metabolic factors.IMPORTANCEColonization of the nasopharynx byStreptococcus pneumoniaeis a precursor to both disease and bacterial transmission. This study challenges the assumption that virulence factors have primarily evolved to cause disease, proposing instead that these pathogenic factors more aligned with facilitating bacterial carriage and spread. Using a pneumococcal clinical isolate strain and two biosynthesis gene mutants in experimental human pneumococcal challenge and epithelial cell line models, we demonstrate that epithelial microinvasion can occur independently of disease causation. Moreover, we uncover a complex relationship between bacterial adaptation and epithelial immune response, with differential effects on epithelial cell reorganization, inflammation, and bacterial growth dynamics. Our findings underscore our contention that pneumococcal colonisation and disease outcomes cannot be predicted by individual gene profiles alone. This phenotyping approach offers the opportunity to effectively unravelS. pneumoniaepathogenesis and its adaptation to the nasopharyngeal niche.Graphical AbstractS. pneumoniaecolonisation is characterised by mucus association, epithelial adherence and microinvasion – where the pneumococcus invades the epithelial barrier without causing disease. Microcolonies form at the epithelial surface and pneumococcal transmigration can occur between epithelial cells. Although mutations inS. pneumoniaebiosynthesis genes (ΔproABCandΔfhs) attenuate disease in a murine model, they do not attenuate microinvasion in either experimental human pneumococcal challenge (EHPC) orin vitroin primary and immortalised epithelial cells. These mutations show strain-dependent effects on both the epithelial and bacterial responses to infection. While not directly co-correlated, factors such as epithelial cellular reorganisation, inflammation and caspase 8 activity alongside pneumococcal metabolic adaptation, virulence factor expression and response to stress are key determinants of the outcome of colonisation in the nasopharyngeal niche.