Biology subjects

Weight, C. M.

Publications and source records attributed to Weight, C. M..

3 recordsLinked to original sources

bioRxiv2023

Bacterial surface lipoproteins mediate epithelial microinvasion by Streptococcus pneumoniae

Streptococcus pneumoniae, a common coloniser of the upper respiratory tract, invades nasopharyngeal epithelial cells without causing disease in healthy people. We hypothesised that surface expression of pneumococcal lipoproteins, recognised by the innate immune receptor TLR2, mediate epithelial microinvasion. Mutation of lgt in serotype 4 (TIGR4) and serotype 6B (BHN418) pneumococcal strains abolishes the ability of the mutants to activate TLR2 signalling. Loss of lgt also led to concomitant decrease in interferon signalling triggered by the bacterium. However, only BHN418 lgt::cm but not TIGR4 lgt::cm was significantly attenuated in epithelial adherence and microinvasion compared to their respective wild-type strains. To test the hypothesis that differential lipoprotein repertoires in TIGR4 and BHN418 lead to the intraspecies variation in epithelial microinvasion, we employed a motif-based genome analysis and identified an additional 525 a.a. lipoprotein (pneumococcal accessory lipoprotein A; palA) encoded by BHN418 that is absent in TIGR4. The gene encoding palA sits within a putative genetic island present in [~]10% of global pneumococcal isolates. While palA was enriched in carriage and otitis media pneumococcal strains, neither mutation nor overexpression of the gene encoding this lipoprotein significantly changed microinvasion patterns. In conclusion, mutation of lgt attenuates epithelial inflammatory responses during pneumococcal-epithelial interactions, with intraspecies variation in the effect on microinvasion. Differential lipoprotein repertoires encoded by the different strains do not explain these differences in microinvasion. Rather, we postulate that post-translational modifications of lipoproteins may account for the differences in microinvasion. IMPORTANCEStreptococcus pneumoniae (pneumococcus) is an important mucosal pathogen, estimated to cause over 500,000 deaths annually. Nasopharyngeal colonisation is considered a necessary prerequisite for disease, yet many people are transiently and asymptomatically colonised by pneumococci without becoming unwell. It is therefore important to better understand how the colonisation process is controlled at the epithelial surface. Controlled human infection studies revealed the presence of pneumococci within the epithelium of healthy volunteers (microinvasion). In this study, we focused on the regulation of epithelial microinvasion by pneumococcal lipoproteins. We found that pneumococcal lipoproteins induce epithelial inflammation but that differing lipoprotein repertoires do not significantly impact the magnitude of microinvasion. Our results highlight the potential importance of the post-translational modification of lipoproteins in the mediation of epithelial invasion during pneumococcal colonisation. Targeting mucosal innate immunity and epithelial microinvasion alongside the induction of an adaptive immune response may be effective in preventing pneumococcal colonisation and disease.

microbiology↗

bioRxiv2023

Impact of Streptococcus pneumoniae biosynthesis gene mutations on epithelial microinvasion and cellular responses

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 ({Delta}proABC/pia and{Delta} fhs/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{Delta} fhs/piaA strain, but not the{Delta} proABC/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. Author SummaryStreptococcus 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 in human and mouse models, 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 AbstractS. 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 ({Delta}proABC and{Delta} fhs) attenuate disease in a murine model, they do not attenuate microinvasion in either experimental human pneumococcal challenge (EHPC), ex vivo or in vitro 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. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=86 SRC="FIGDIR/small/545009v8_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@11a17aeorg.highwire.dtl.DTLVardef@8c707org.highwire.dtl.DTLVardef@42abf8org.highwire.dtl.DTLVardef@12850f9_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2018

Epithelial control of colonisation by Streptococcus pneumoniae at the human mucosal surface

Control of Streptococcus pneumoniae colonisation at human mucosal surfaces is critical to reducing the burden of pneumonia and invasive disease, interrupting onward transmission, and in achieving herd protection. We hypothesised that the pattern of pneumococcal-epithelial engagement dictates the inflammatory response to colonisation, and that this epithelial sensing is linked to bacterial clearance. Here we have used nasal curette biopsies from a serotype 6B Experimental Human Pneumococcal Carriage Model (EHPC) to visualize S. pneumoniae colonisation and relate these interactions to epithelial surface marker expression and transcriptomic profile upregulation. We have used a Detroit 562 cell co-culture model to further understand these processes and develop an integrated epithelial transcriptomic module to interrogate gene expression in the EHPC model. We have shown for the first time that pneumococcal colonisation in humans is characterised by microcolony formation at the epithelial surface, microinvasion, cell junction protein association, epithelial sensing, and both epithelial endocytosis and paracellular transmigration. Comparisons with other clinical strains in vitro has revealed that the degree of pneumococcal epithelial surface adherence and microinvasion determines the host cell surface marker expression (ICAM-1 and CD107), cytokine production (IL-6, IL-8 and ICAM-1) and the transcriptomic response. In the context of retained barrier function, epithelial microinvasion is associated with the upregulation of a wide range of epithelial innate signalling and regulatory pathways, inflammatory mediators, adhesion molecules, cellular metabolism and stress response genes. The prominence of epithelial TLR4R signalling pathways implicates pneumolysin, a key virulence factor, but although pneumolysin gene deletion partially ameliorates the inflammatory transcriptional response in vitro, critical inflammatory pathways persist in association with enhanced epithelial adhesion and microinvasion. Importantly, the pattern of the host-bacterial interaction seen with the 6B strain in vitro is also reflected in the EHPC model, with evidence of microinvasion and a relatively silent epithelial transcriptomic profile that becomes most prominent around the time of bacterial clearance. Together these data suggest that epithelial sensing of the pneumococcus during colonisation in humans is enhanced by microinvasion, resulting in innate epithelial responses that are associated with bacterial clearance.\n\nHighlights\n\nO_LIColonisation of the human mucosa by Streptococcus pneumoniae is associated with microcolony formation, microinvasion, epithelial sensing and an epithelial innate response.\nC_LIO_LIFollowing adherence to the epithelial cell surface, microinvasion of the epithelium may occur by endocytosis and/or lateral migration between cells without necessarily compromising barrier integrity.\nC_LIO_LIThe pattern of pneumococcal epithelial surface adherence and microinvasion determines the host cell response through a range of innate signaling and regulatory pathways, inflammatory mediators, adhesion molecules, cellular metabolism and stress response genes.\nC_LIO_LIEpithelial sensing is triggered by, but not wholly dependent on pneumolysin, a key virulence factor of S. pneumoniae.\nC_LI

immunology↗