Michael G. Connor

NF-κB driven cellular immunity is essential for both pro- and anti-inflammatory responses to microbes, which makes it one of the most frequently targeted pathways by bacteria during pathogenesis. How NF-κB tunes the epithelial response to Streptococcus pneumoniae across the spectrum of commensal to pathogenic outcomes is not fully understood. In this study, we compare a commensal-like 6B ST90 strain to an invasive TIGR4 strain and demonstrate, through comparative mass spectrometry of the p65 interactome, that TIGR4 challenge triggers interaction of COMMD2 with p65 and p62. Mechanistically, we show this complex mediates export of p65 for degradation and COMMD2 is necessary for altering host cellular immunity. With these results, we reveal a bacterial pathogenesis mechanism to repress host inflammatory response though COMMD2 and p65 degradation while presenting a paradigm for diverging NF-κB responses to pneumococcus.

Background: Nasopharyngeal colonization by Streptococcus pneumoniae is characterized by bacterial adherence to epithelial cells, microinvasion, and innate immune activation. Previously, we have shown that two serotype 6B S pneumoniae mutant strains affecting bacterial metabolism (ΔproABC/pia and Δfhs/pia) colonize humans and mice, but in a murine disease model do not cause invasive infection. Methods: Using an experimental human pneumococcal challenge model, ex vivo airway cells, and in vitro nasopharyngeal epithelium, we explore whether microinvasion and innate immune responses persist despite disease attenuation. Results: 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 wild-type. Cellular reorganization of primary human airway epithelium varied considerably between strains. Compared to wild-type, infection of Detroit 562 epithelial cells by the Δfhs/piaA strain, but not the ΔproABC/piaA strain, was less proinflammatory, induced less caspase 8 production, and was associated with increased pneumococcal hydrogen peroxide and reduced pneumolysin secretion. Conclusions: These findings suggest that differences in microinvasion and epithelial responses were driven by the differential expression of multiple bacterial virulence and metabolic pathways. These data highlight the complex impact of single gene mutations on bacterial virulence and suggest that the virulence determinants of pneumococcal epithelial colonization, microinvasion, and innate immunity are not necessarily directly linked to disease.

Crosstalk between respiratory bacteria and human airway epithelial cells initiates cellular immunity, yet the molecular determinants of asymptomatic carriage versus inflammation remain incompletely understood. A key regulator of airway epithelial cellular immunity is the Signal Transducer and Activator of Transcription (STAT3). Once activated by phosphorylation this host transcription factor signals through two cascades, canonical by phosphorylation at tyrosine 705 and non-canonical by phosphorylation at serine 727, which contributes to bacterial clearance and immune defense in pneumonia. Despite this, its role in epithelial responses to Streptococcus pneumoniae has not been defined. Revealing these processes are particularly important for understanding the variability in pathogenic potential for the pathobiont Streptococcus pneumoniae which triggers either host commensal-like carriage or susceptibility at this interface. Here we investigated the role of STAT3 signaling during pneumococcal challenge using lower airway epithelial cells and two pneumococcal isolates favoring either commensal-like carriage, 6B ST90, or with pathogenic potential, TIGR4. We show the invasive TIGR4 strain drives canonical STAT3 phosphorylation and suppresses non-canonical STAT3 in a burden–dependent manner. This contrasts with the 6B ST90 isolate which requires a 20-fold increase in burden to elicit minimal STAT3 responses. TIGR4 activation correlated with SOCS3 expression, while knockdown of endogenous STAT3 did not alter pneumococcal adherence or epithelial membrane integrity. Mechanistically, STAT3 activation required live bacteria and pneumolysin production but was independent of bacterial hydrogen peroxide. Altogether we reveal previously unrecognized divergence in STAT3 signaling during pneumococcal infection.

In this study, we developed novel lung organoid-on-chip models that elucidate differential human tissue response to various strains of respiratory pathogens: Streptococcus pneumoniae and SARS-CoV-2. We show that human fetal-derived distal lung epithelial cells are readily expandable in 3D as organoids, thereby providing a highly sustainable source of lung progenitor cells. These 3D organoid progenitors can then be induced to produce airway and alveolar organoids on microfluidic devices. Upon challenge with Streptococcus pneumoniae , a bacterium known to cause pneumonia, a rapid and strain-dependent colonization was observed at the epithelial surface of alveolar chips. We also assessed SARS-CoV-2 infection in the alveoli-on-chip system and observed that the Delta variant exhibited greater infectivity as compared to the Omicron BA.5. Both SARS-CoV-2 variants induced potent interferon responses and triggered the expression of different interferon-stimulated genes. Our results demonstrate that strain-specific host defense mechanisms can be recapitulated in human-organoid-based microfluidic systems, paving the way for the use of such platforms for more targeted assessments of human response to novel emergent pathogen strains. Highlights: Human fetal epithelial lung stem cells can be expanded as multipotent organoids and differentiated into both airway or alveolar organoids Multipotent lung organoids efficiently produce functional epithelia of small airway or alveoli when grown on-chip. Streptococcus pneumoniae inoculation in alveoli-on-chip mimics the early stages of bacterial colonization in lung epithelia Alveoli on-chip system recapitulates variant-specific interactions. SARS-CoV-2 Delta replicates but not Omicron BA.5. Robust interferon response upon SARS-CoV-2 infection shows Alveoli on-chip can model innate immune responses.

NF-κB driven cellular immunity is essential for both pro- and anti-inflammatory responses to microbes, which makes it one of the most frequently targeted pathways by bacteria during pathogenesis. How NF-κB tunes the epithelial response to Streptococcus pneumoniae across the spectrum of commensal to pathogenic outcomes is not fully understood. In this study, we compare a commensal-like 6B ST90 strain to an invasive TIGR4 isolate and demonstrate that TIGR4 both blunts and antagonizes NF-κB activation. We identified, through comparative mass spectrometry of the p65 interactome, that the 6B ST90 isolate drives a non-canonical NF-κB RelB cascade, whereas TIGR4 induces p65 degradation though aggrephagy. Mechanistically, TIGR4 challenge triggers a novel interaction of COMMD2 with p65 and p62. This complex mediates export of p65 for degradation and is necessary for repressing host inflammatory transcription. With these results, we reveal for the first time a new bacterial pathogenesis mechanism to repress host inflammatory response though COMMD2 mediated turnover of p65, and present a paradigm for diverging NF-κB responses to pneumococcus.

Epithelial cells are the first point of contact for bacteria entering the respiratory tract. Streptococcus pneumoniae is an obligate human pathobiont of the nasal mucosa, carried asymptomatically but also the cause of severe pneumonia. The role of the epithelium in maintaining homeostatic interactions or mounting an inflammatory response to invasive S. pneumoniae is currently poorly understood. However, studies have shown that chromatin modifications, at the histone level, induced by bacterial pathogens interfere with the host transcriptional program and promote infection. In this study, we demonstrate that S. pneumoniae actively induces di-methylation of lysine 4 on histone H3 (H3K4me2), which persists for at least 9 days upon clearance of bacteria with antibiotics. We show that infection establishes a unique epigenetic program affecting the transcriptional response of epithelial cells, rendering them more permissive upon secondary infection. Our results establish H3K4me2 as a unique modification induced by infection, distinct from H3K4me3, which localizes to enhancer regions genome-wide. Therefore, this study reveals evidence that bacterial infection leaves a memory in epithelial cells after bacterial clearance, in an epigenomic mark, thereby altering cellular responses for subsequent infection.

NF-κB driven cellular immunity is essential for both pro- and anti-inflammatory responses to microbes, which makes it one of the most frequently targeted pathways by bacteria during pathogenesis. How NF-κB tunes the epithelial response to Streptococcus pneumoniae across the spectrum of commensal to pathogenic outcomes is not fully understood. In this study, we compare a commensal-like 6B ST90 strain to an invasive TIGR4 isolate and demonstrate, through comparative mass spectrometry of the p65 interactome, TIGR4 challenge triggers a novel interaction of COMMD2 with p65 and p62. Mechanistically, we show this complex mediates export of p65 for degradation and COMMD2 is necessary for altering host cellular immunity. With these results, we reveal for the first time a new bacterial pathogenesis mechanism to repress host inflammatory response though COMMD2 and p65 degradation while presenting a paradigm for diverging NF-κB responses to pneumococcus.

Streptococcus pneumoniae is a natural colonizer of the human respiratory tract and an opportunistic pathogen. Although epithelial cells are among the first to encounter pneumococci, the cellular processes and contribution of epithelial cells to the host response are poorly understood. Here, we show that a S. pneumoniae serotype 6B ST90 strain, which does not cause disease in a murine infection model, induces a unique NF-κB signature response distinct from an invasive-disease-causing isolate of serotype 4 (TIGR4). This signature is characterized by activation of p65 and requires a histone demethylase KDM6B. We show, molecularly, that the interaction of the 6B strain with epithelial cells leads to chromatin remodelling within the IL-11 promoter in a KDM6B-dependent manner, where KDM6B specifically demethylates histone H3 lysine 27 dimethyl. Remodelling of the IL-11 locus facilitates p65 access to three NF-κB sites that are otherwise inaccessible when stimulated by IL-1β or TIGR4. Finally, we demonstrate through chemical inhibition of KDM6B with GSK-J4 inhibitor and through exogenous addition of IL-11 that the host responses to the 6B ST90 and TIGR4 strains can be interchanged both in vitro and in a murine model of infection in vivo. Our studies therefore reveal how a chromatin modifier governs cellular responses during infection.