Biology subjects

Venturini, C.

Publications and source records attributed to Venturini, C..

18 recordsLinked to original sources

bioRxiv2025

Microenvironmental TGF-β is an early driver of NF1-associated tumour formation

Neurofibromatosis Type 1 (NF1) is a common tumour predisposition syndrome characterised by neurofibromas - Nf1-/- Schwann cell (SC)-derived tumours of peripheral nerves. We and others have shown that Nf1 loss in SCs is insufficient for neurofibroma formation but cooperates with an injury microenvironment to form tumours, but the mechanisms remained unknown. Here, we identify macrophage-secreted TGF-{beta} as the microenvironmental injury signal that is essential for tumourigenesis. Analysis of the earliest stages of neurofibroma formation showed that tumours arise from a population of Nf1-/- SCs that escape the regenerating nerve shortly after injury. Here, they reside in a distinct microenvironment conducive for tumourigenesis, where TGF-{beta} disrupts SC/axonal interactions and SC re-differentiation. Pharmacological inhibition of TGF-{beta} for a short therapeutic window during this early stage inhibited tumour formation, highlighting the potential to normalise Nf1-/- SCs and identifying TGF-{beta} as a potential therapeutic target to both treat and prevent neurofibroma formation.

cell biology↗

bioRxiv2025

Vibrio harveyi plasmids as drivers of virulence in barramundi (Lates calcarifer)?

Vibrio species are an emerging public and animal health risk in marine environments and the opportunistic bacterial pathogen Vibrio harveyi is a major disease risk for tropical aquaculture. Current understanding of virulence in V. harveyi is limited by strain-specific variability and complex host-pathogen dynamics. This study sought to integrate genomic investigation, phenotypic characterisation and in vivo challenge trials in barramundi (Lates calcarifer) to increase our understanding of V. harveyi virulence. We identified two hypervirulent isolates, Vh-14 and Vh-15 that caused 100% mortality in fish within 48 hours, and that were phenotypically and genotypically distinct from other V. harveyi isolates. Virulent isolates contained multiple plasmids, including a 105,412 bp conjugative plasmid with type III secretion system genes originally identified in Yersinia pestis. The emergence of this hypervirulent plasmid-mediated patho-variant poses a potential threat to the sustainable production of marine finfish in Southeast Asia, the Mediterranean and Australia. In addition, we observed an effect of temperature on phenotypic indicators of virulence with an increase in activity at 28{degrees}C and 34{degrees}C compared to 22{degrees}C. This suggests that temperature fluctuations associated with climate change may act as a stressor on bacteria, increasing virulence gene secretion and host adaptation. Our results utilising a myriad of technologies and tools, highlights the importance of a holistic view to virulence characterisation.

microbiology↗

bioRxiv2024

Single-cell transcriptome and T cell receptor profiling of the tuberculin skin test

The tuberculin skin test (TST) is a cutaneous delayed hypersensitivity reaction to antigen from Mycobacterium tuberculosis (Mtb). We provide the first single cell sequencing characterisation of the human TST reaction, based on skin suction blisters induced at the site of the TST on day 2 in 31 individuals. Integrated single cell RNA and TCR sequencing showed the immune response to be dominated by T cells, with smaller populations of NK cells and myeloid cells. T cells comprised CD4, CD8, gamma/delta and NK T cells, with 50% of all T cells identified as cytotoxic and 14% as regulatory. Interferon gamma gene expression was strongest in CD8 T cells, and distinct CD4 T helper lineages could not unambiguously be identified at this time point. Amongst myeloid cells, 63% displayed antimicrobial gene expression and 28% were functionally polarised towards antigen presentation with higher levels of HLA class 2 expression. We derived and validated transcriptional signatures for cell types and cellular functions relevant to the immune landscape of the TST. These data help to improve our understanding of the immune response to Mtb and enable further exploration of bulk transcriptomic data through context-specific cellular deconvolution.

immunology↗

bioRxiv2024

The CSF transcriptome in pneumococcal meningitis reveals compartmentalised host inflammatory responses associated with mortality.

AbstractO_ST_ABSBackgroundC_ST_ABSPneumococcal meningitis (PM) has persistently poor clinical outcomes, especially in sub- Saharan Africa. To better characterise the inflammatory response and identify factors associated with mortality we compared paired peripheral blood and cerebrospinal fluid (CSF) transcriptomes before the initiation of antibiotics in Malawian adults with proven PM. ResultsBlood transcriptional profiles were obtained in 28 patients with PM, with simultaneous paired with CSF profiles available for 13 patients. 15/28 (52%) patients died. Comparison of the transcriptome between CSF and blood compartments showed upregulation of 2293 differentially expressed genes in CSF and 909 in blood; enriched pathways in CSF included inflammasome activity and neutrophil migration/activation in the CSF, contrasting with enrichment for pathways including platelet and endothelial activation, cell cycle, cytokine release and oxidative stress in the blood transcriptome. Comparison of CSF profiles between survivors and non-survivors revealed 1829 differentially expressed genes, non- survivor CSF was enriched for multiple innate inflammatory pathways, including IL-17A and Type 1 interferons and proteolysis. In contrast, minimal transcriptomic differences between outcome groups were detected in blood. ConclusionInflammation in PM is characterised by compartmentalised responses in blood and CSF. Poorer outcomes are associated with an dysregulated innate immune host response to S. pneumoniae in the CSF compartment.

immunology↗

bioRxiv2023

A multi-scale clutch model for adhesion complex mechanics

Cell-matrix adhesion is a central mechanical function to a large number of phenomena in physiology and disease, including morphogenesis, wound healing and tumor cell invasion. Today, how single cells responds to different extracellular cues has been comprehensibly studied. However, how the mechanical behavior of the main individual molecules that form an adhesion complex cooperatively respond to force within the adhesion complex has not been addressed. This is a key aspect in cell adhesion because how these cell adhesion molecules respond to force determines not only cell-matrix behavior but, ultimately, cell function. To answer this question, we develop a multi-scale computational model for adhesion complexes mechanics. Based on the classical clutch hypothesis, we model individual adhesion chains made of a contractile actin network, a talin rod and an integrin molecule that binds at individual adhesion sites on the extracellular matrix. We explore several scenarios of integrins dynamics and analyze the effects of diverse extracellular matrices on the behavior of the adhesion molecules and on the whole adhesion complex. Our results explains how every single component of the adhesion chain mechanically responds to the contractile actomyosin force and show how they control the tractions forces exerted by the cell on the extracellular space. Importantly, our computational results are in agreement with previous experimental data both at the molecular and cell level. Our multi-scale clutch model presents a step forward not only to further understand adhesion complexes mechanics but also to, e.g., engineer better biomimetic materials, repair biological tissues or arrest invasive tumor migration. Author summaryCell-matrix adhesions are directly implicated in key biological processes such as tissue development, regeneration and tumor cell invasion. These cell functions are determined by how adhesion complexes feel and respond to mechanical forces. Still, how forces are transmitted through the individual cell adhesion molecules that integrate the adhesion complex is poorly understood. To address this issue, we develop a multi-scale clutch model for adhesion complexes where individual adhesion chains, made of integrin and talin molecules, are considered within classical clutch models. This approach provides a rich mechanosensivity insight of how the mechanics of cell adhesion works. It allows to integrate accurate biophysical models of individual adhesion molecules into whole adhesion complex models. Our multi-scale clutch approach allows to extend our current knowledge of adhesion complexes for physiology and disease, e.g., the regeneration of biological tissues or arrest invasive tumor migration, and for engineering better biomimetic materials.

biophysics↗

bioRxiv2022

A full computational model of cell motility: Early spreading, cell migration and competing taxis

Cell motility represents one of the most fundamental function in mechanobiology. Cell motility is directly implicated in development, cancer or tissue regeneration, but it also plays a key role in the future of tissue and biomedical engineering. Here, we derived a computational model of cell motility that incorporates the most important mechanisms toward cell motility: cell protrusion, polarization and retrograde flow. We first validate our model to explain two important types of cell migration, i.e. confined and ameboid cell migration, as well as all phases of the latter cell migration type, i.e. symmetric cell spreading, cell polarization and latter migration. Then, we use our model to investigate durotaxis and chemotaxis. The model predicts that chemotaxis alone induces larger migration velocities than durotaxis and that durotaxis is activated in soft matrices but not in stiff ones. More importantly, we analyze the competition between chemical and mechanical signals. We show that chemotaxis rules over durotaxis in most situations although durotaxis diminishes chemotaxis. Moreover, we show that inhibiting the effect of GTPases in actin polymerization at the cell front may allow durotaxis to take control over chemotaxis in soft substrates. Understanding how the main forces in cell motility cooperate, and how a precise manipulation of external cues may control directed cell migration is not only key for a fundamental comprehension of cell biology but also to engineer better biomimetic tissues. To this end, we provide a freely-available platform to predict all phases and modes of cell motility analyzed in this work.

biophysics↗

bioRxiv2022

Positive, negative and engineered durotaxis

Cell migration is a physical process central to life. Among others, it regulates embryogenesis, tissue regeneration and tumor growth. Therefore, understanding and controlling cell migration represent fundamental challenges in science. Specifically, the ability of cells to follow stiffness gradients, known as durotaxis, is ubiquitous across most cell types. Even so, certain cells follow positive stiffness gradients while others move along negative gradients. How the physical mechanisms involved in cell migration works to enable a wide range of durotactic responses is still poorly understood. Here, we provide a mechanistic rationale of durotaxis by integrating stochastic clutch models for cell adhesion with an active gel theory of cell migration. We show that positive and negative durotaxis found across cell types are explained by asymmetries in the cell adhesion dynamics. We rationalize durotaxis by an asymmetric mechanotransduction in the cell adhesion behavior that further polarizes the intracellular retrograde flow and the protruding velocity at the cell membrane. Our theoretical framework confirms previous experimental observations and explains positive and negative durotaxis. Moreover, we show how durotaxis can be engineer to manipulate cell migration, which has important implications in biology, medicine and bioengineering.

biophysics↗

bioRxiv2021

L-form switching confers antibiotic, phage and stress tolerance in pathogenic Escherichia coli

The bacterial L-form is induced by exposure to cell wall targeting antibiotics or innate immune effectors such as lysozyme and is likely to be important in many human infections. Here, we demonstrate that the osmotically fragile L-form is a distinct physiological state in Escherichia coli that is highly tolerant of oxidative stress and resistant to powerful antibiotics and common therapeutic bacteriophages. L-forms quickly revert (<20h) to their cell-walled state after antibiotic withdrawal, with apparently normal physiology and with few or no changes in DNA sequence. T4-like phages that are obligately lytic in cell-walled E. coli preferentially pseudolysogenise their L-forms providing them with transient superinfection immunity. Our data indicate that L-form switching is a common response of pathogenic E. coli strains to cell wall-targeting antibiotics and that the most commonly used lytic bacteriophages are ineffective against them in this state.

microbiology↗

bioRxiv2020

Differential effects of antibiotic treatment with piperacillin/tazobactam or ceftriaxone on the murine gut microbiota

Effective antimicrobial stewardship requires a better understanding of the impact of different antibiotics on the gut microflora. Studies in humans are confounded by large inter-individual variability and difficulty in identifying control cohorts. However, controlled murine models can provide valuable information. We examined the impact of a penicillin-like antibiotic (piperacillin/tazobactam, TZP) or a third-generation cephalosporin (ceftriaxone, CRO) on the murine gut microbiota. We analyzed gut microbiome composition by 16S-rRNA amplicon sequencing and effects on the Enterobacteriaceae by qPCR and standard microbiology. Colonization resistance to multidrug resistant Escherichia coli ST131 and Klebsiella pneumoniae ST258 was also tested. Changes in microbiome composition and a significant (p<0.001) decrease in diversity occurred in all treated mice, but were more marked and longer lasting after CRO exposure with a persistent rise in Proteobacteria levels. Increases in the Enterobacteriaceae occurred in all antibiotic treated mice, but were transient and associated with direct antibiotic pressure. Co-habitation of treated and untreated mice attenuated the detrimental effect of antibiotics on treated animals, but also caused disturbance in untreated co-habitants. At the height of dysbiosis after antibiotic termination, the murine gut was highly susceptible to colonization with both multidrug resistant pathogens. The administration of a third-generation cephalosporin caused a significantly prolonged dysbiosis in the murine gut microflora, when compared to a penicillin/{beta}-lactam inhibitor combination with comparable activity against medically important virulent bacteria. At the height of dysbiosis, both antibiotic treatments equally led to microbial imbalance associated with loss of resistance to gut colonization by antibiotic-resistant pathogens.

microbiology↗

bioRxiv2020

cmvdrg - An R package for Human Cytomegalovirus antiviral Drug Resistance Genotyping

The prevention and treatment of many herpesvirus associated diseases is based on the utilization of antiviral therapies, however therapeutic success is limited by the development of drug resistance. A comprehensive point of truth of resistance conferring mutations has been missing but would be important to aid the development of antiviral drugs and in management of infections. We therefore developed HerpesDRG, a drug resistance mutation database for all the known important genes and current treatment options, built from a systematic review of available genotype to phenotype literature. The database is released along with an R package to provide a low barrier of entry to variant resistance annotation and clinical implication analysis from common sanger and NGS sequence data. This represents the first openly available and community maintainable knowledgebase of drug resistance mutations for the human herpes viruses (HHV), developed for the community of researchers and clinicians tackling HHV drug resistance. AvailabilityThe HerpesDRG database is available at github.com/ojcharles/herpesdrg-db. The R package for resistance genotyping data is available at github.com/ojcharles/herpesdrg. A user-friendly webserver is available at cmv-resistance.ucl.ac.uk/herpesdrg

bioinformatics↗

bioRxiv2019

Concise whole blood transcriptional signatures for incipient tuberculosis: A systematic review and patient-level pooled meta-analysis

Blood transcriptional signatures may predict risk of tuberculosis (TB). We compared the performance of 17 mRNA signatures in a pooled dataset comprising 1,026 samples, including 183 samples from 127 incipient TB cases, from four studies conducted in South Africa, Ethiopia, The Gambia and the UK. We show that eight signatures (comprising 1-25 transcripts) that predominantly reflect interferon inducible gene expression, have equivalent diagnostic accuracy for incipient TB over a two-year period with areas under the receiver operating characteristic curves ranging from 0.70 (95% confidence interval 0.64-0.76) to 0.77 (0.71-0.82). The sensitivity of all eight signatures declined with increasing disease-free time interval. Using a threshold derived from two standard deviations above the mean of uninfected controls giving specificities of >90%, the eight signatures achieved sensitivities ranging 24.7-39.9% over a 24 month interval, rising to 47.1-81.0% over 3 months. Based on pre-test probability of 2%, the eight signatures achieved positive predictive value ranging from 6.8-9.4% over 24 months, rising to 11.1-14.3% over 3 months. When using biomarker thresholds maximising sensitivity and specificity with equal weighting to both, no signature met the minimum World Health Organization (WHO) Target Product Profile parameters for incipient TB biomarkers over a two-year period. Blood transcriptional biomarkers reflect short-term risk of TB and only exceed WHO benchmarks if applied to 3-6 month intervals.

microbiology↗

bioRxiv2019

K. pneumoniae ST258 genomic variability and bacteriophage susceptibility

Multidrug resistant carbapenemase-producing Klebsiella pneumoniae capable of causing severe disease in humans is classified as an urgent threat by health agencies worldwide. Bacteriophages are being actively explored as potential therapeutics against these multidrug resistant pathogens. We are currently developing bacteriophage therapy against carbepenem-resistant K. pneumoniae belonging to the genetically diverse, globally disseminated clonal group CG258. In an effort to define a robust experimental approach for effective selection of lytic viruses for therapy, we have fully characterized the bacterial genomes of 18 target strains, tested them against novel lytic bacteriophages, and generated phage-susceptibility profiles. The genomes of K. pneumoniae isolates carrying blaNDM and blaKPC were sequenced and isolates belonging to CG258 were selected for susceptibility testing using a panel of lytic bacteriophages (n=65). The local K. pneumoniae CG258 population was dominated by isolates belonging to sequence type ST258 clade 1 (86%). The primary differences between ST258 genomes were variations in the capsular locus (cps) and in prophage content. We showed that CG258-specific lytic phages primarily target the capsule, and that successful infection is blocked in many, post-adsorption, by immunity conferred by existing prophages. Five bacteriophages specifically active against K. pneumoniae ST258 clade 1 (n=5) belonging to the Caudovirales order were selected for further characterization. Our findings show that effective control of K. pneumoniae CG258 with phage will require mixes of diverse lytic viruses targeting all relevant cps variants and allowing for variable prophage content. These insights will facilitate identification and selection of therapeutic phage candidates against this serious pathogen.\n\nImportanceBacteriophages are natural agents that exclusively and selectively kill bacteria and have the potential to be useful in the treatment of multidrug resistant infections. K. pneumoniae CG258 is a main agent of life-threatening sepsis that is often resistant to last-line antibiotics. Our work highlights some key requirements for developing bacteriophage preparations targeting this pathogen. By defining the genomic profile of our clinical K. pneumoniae CG258 population and matching it with bacteriophage susceptibility patterns, we found that bacteriophage ability to lyse each strain correlates well with K. pneumoniae CG258 structural subtypes (capsule variants). This indicates that preparation of bacteriophage therapeutics targeting this pathogen should aim at including phages against each bacterial capsular subtype. This necessitates a detailed understanding of the diversity of circulating isolates in different geographical areas in order to make rational therapeutic choices.

microbiology↗

bioRxiv2019

Both the inflammatory response and clinical outcome differ markedly between adults with pneumococcal and meningococcal meningitis in a high HIV-1 prevalent setting in sub-Saharan Africa

Outcomes from pneumococcal meningitis (PM) are worse than meningococcal meningitis (MM), particularly in settings with high HIV-1 prevalence, but the reasons are unknown. We compared inflammatory responses between PM and MM in Malawian adults. As compared to MM (n=27, 67% HIV-infected, mortality 11%), patients with PM (n=440, 84% HIV-infected, mortality 54%) were older, had strikingly lower CSF WCC, higher pro-inflammatory cytokine concentrations and higher mortality. PM is characterized by significantly lower CSF WCC, but greater inflammation and higher mortality compared to MM. Mechanistic understanding of blunting of the CSF leukocyte response in PM in-vivo is required.

immunology↗

bioRxiv2019

Exaggerated in vivo IL-17 responses discriminate recall responses in active TB

Host immune responses at the site of Mycobacterium tuberculosis (Mtb) infection serve to contain the pathogen, but also mediate the pathogenesis of tuberculosis (TB) and onward transmission of infection. Interferon gamma (IFN{gamma}) responses do not discriminate between protection and pathogenicity, but IL-17A/F responses, known to drive pathology in diverse chronic inflammatory diseases, have also been associated with TB pathogenesis in animal models. At the site of in vivo immune recall responses to Mtb modelled by the tuberculin skin test, we show for the first time that active TB in humans is also associated with exaggerated IL-17A/F expression, accumulation of Th17 cells and IL-17A/F bioactivity, including increased neutrophil recruitment and matrix metalloproteinase-1 expression directly implicated in TB pathogenesis. These features discriminate recall responses in patients with active TB from those with cured or latent infection and are also evident at the site of TB disease. Our data support targeting of this pathway in host-directed therapy for TB.

immunology↗

bioRxiv2018

Cerebrospinal fluid transcriptional analyses reveals upregulation of IL-17, Type 1 interferon transcriptional pathways and neutrophil persistence genes associated with increased mortality from pneumococcal meningitis in adults

BackgroundImproving outcomes from pneumococcal meningitis (PM), particularly in populations with high HIV prevalence, requires better understanding of host inflammatory responses to infection.\n\nMethodsWe compared the transcriptome in pre-antibiotic cerebrospinal fluid (CSF) and blood from Malawian adults with PM using RNA sequencing. We used network analyses and cellular/process deconvolution of the transcriptome to identify important patho-physiological associations with outcome.\n\nFindingsBlood transcriptional profiles were obtained in 28 patients (21 HIV co-infected; median age 33 years [26-66]; median CSF WCC 28 cells/mm3 [0-3660]; median bacterial load 4.7x106 copies/ml CSF [671-2x109]; in-hospital mortality 64%), paired CSF profiles were obtained in 13. Marked differences in gene expression by outcome were confined to the CSF. In non-survivors, differentially expressed genes in the CSF were co-correlated in a network of pro-inflammatory gene-clusters enriched for collagen degradation and platelet degranulation. In contrast, CSF gene expression networks from surviving patients were dominated by DNA repair, transcriptional regulation and immunological signalling. CSF expression of gene response-modules for IL-17, Type 1 interferons and IL-10 were enriched in non-survivors, expression of cell-specific response-modules did not differ by outcome. However, genes for neutrophil chemotaxis and persistence were highly over-expressed in non-survivors.\n\nInterpretationThese data suggest poor outcome in PM is associated with over-expression of IL-17 and T1-IFN associated pro-inflammatory responses in the CSF and suggest a role for neutrophil-mediated inflammation. These responses are unlikely to be effected by current adjunctive treatments. Improving poor outcomes from PM will require better-targeted interventions.\n\nFundingAcademy of Medical Sciences (UK), Wellcome Trust (UK) (089671/B/09/Z)

immunology↗

bioRxiv2018

Immune profiling of cord blood after prolonged rupture of membranes.

We hypothesised that foetal immune responses to an infectious challenge may be detected by genome-wide transcriptional profiling of cord blood. In order to test this hypothesis, we sought to identify transcriptomic changes in post-natal cord blood samples following prolonged pre-labour rupture of membranes (PROM) as a surrogate for increased risk of infection. By comparison to controls we found increased levels of blood transcripts in a subset of prolonged PROM cases, significantly enriched for innate immune system signalling pathways. These changes were idiosyncratic, suggesting qualitative and quantitative variation in foetal immune responses which may reflect differences in exposure and/or in host genetics. Our data support the view that PROM represents an infection risk to the foetus. In addition, we propose that cord blood transcriptional profiling offers exciting opportunities to identify immune correlates of clinical outcome following potential in utero exposures to infection. These may be used to elucidate the mechanisms of immunological protection and pathology in the foetus and identify biomarkers to stratify the risk of adverse outcomes.

immunology↗

bioRxiv2018

Whole genome analysis of ExPEC ST73 from a single hospital over a 2-year period identified different circulating clonal groups

ST73 has emerged as one of the most frequently isolated extraintestinal pathogenic E. coli (ExPEC). To examine the localised diversity of ST73 clonal groups including their mobile genetic elements profile, we sequenced the genomes of 16 multiple drug-resistant ST73 isolates from patients with urinary tract infection from a single hospital in Sydney, Australia between 2009 and 2011. Genome sequences were used to generate a SNP-based phylogenetic tree to determine the relationship of these isolates in a global context with ST73 sequences (n=210) from public databases. There was no evidence of a dominant outbreak strain of ST73 in patients from this hospital, rather we identified at least eight separate groups, several of which reoccur, over a two-year period. The inferred phylogeny of all ST73 strains (n=226) including the ST73 Clone D i2 reference genome shows high bootstrap support and clusters into four major groups which correlate with serotype. The Sydney ST73 strains carry a wide variety of virulence-associated genes but the presence of iss, pic and several iron acquisition operons was notable.\n\nImpactST73 is a major clonal lineage of ExPEC that causes urinary tract infections often with uroseptic sequelae but has not garnered substantial scientific interest as the globally disseminated ST131. Isolation of multiple antimicrobial resistant variants of ExPEC ST73 have increased in frequency, but little is known about the carriage of class 1 integrons in this sequence type and the plasmids that are likely to mobilise them. This pilot study examines the ST73 isolates within a single hospital in Sydney Australia and provides the first large-scale core-genome phylogenetic analysis of ST73 utilizing public sequence read datasets. We used this analysis to identify at least 8 sub-groups of ST73 within this single hospital. Mobile genetic elements associated with antibiotic resistance were less diverse and only three class 1 integron structures were identified, all sharing the same basic structure suggesting that the acquisition of drug resistance is a recent event. Genomic epidemiological studies are needed to further characterise established and emerging clonal populations of multiple drug resistant ExPEC to identify sources and aid outbreak investigations.

genomics↗

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↗