key: cord-0430077-e9vqvbw0
authors: Kahlert, C. R.; Nigg, S.; Onder, L.; Dijkman, R.; Diener, L.; Rodriguez, R.; Vernazza, P.; Thiel, V.; Vidal, J. E.; Albrich, W. C.
title: The quorum sensing com system regulates pneumococcal colonisation and invasive disease in a pseudo-stratified airway tissue model
date: 2021-12-17
journal: nan
DOI: 10.1101/2021.12.16.21267943
sha: 782809f90195307f6a55b34e791a02255d7bf648
doc_id: 430077
cord_uid: e9vqvbw0

Streptococcus pneumoniae (Spn) colonises respiratory epithelia but can also invade lung cells causing pneumonia. We developed an ex vivo model with human airway epithelial (HAE) cells harvested from lung biopsies to study Spn colonisation and translocation. Flow-cytometry, confocal imaging and electron microscopy studies identified the epithelial lineage with signs of differentiation (beating cilia, mucus, and tight junctions). HAE cells were challenged with Spn wild-type TIGR4 (wtSpn) or its isogenic {Delta}comC quorum sensing-deficient mutant. {Delta}comC mutant colonised significantly less than wtSpn at 6 h post-inoculation but at significantly higher levels at 19 h and 30 h. Translocation correlated inversely with colonisation density. Transepithelial electric resistance (TEER) decreased after pneumococcal infection and correlated with increased translocation for both strains. Confocal imaging illustrated colocalisation of intracellular Spn with both cilia and zonulin-1 and prominent microcolony formation with wtSpn but disintegration of microcolony structures over time with {Delta}comC mutant. {Delta}comC caused a more pronounced release of both zonulin-1 and lactate dehydrogenase into the supernatant at later time points than wtSpn, suggesting that cytotoxicity is likely not the mechanism leading to translocation. There was a density- and time-dependent increase of inflammatory cytokines from human HAE cells infected with {Delta}comC compared with wtSpn, including increased levels of the NLRP3 inflammasome-related IL-18. In conclusion, our experiments indicate that ComC system allows a higher organisational level of population structure resulting in microcolony formation, increased early colonisation and subsequent translocation. We propose that ComC inactivation unleashes a very different and possibly more virulent phenotype that merits further investigation.

Community-acquired pneumonia due to Streptococcus pneumoniae (Spn) is associated with high morbidity and mortality worldwide [1] . Currently there are at least 100 different pneumococcal serotypes known with different ability to cause mucosal and invasive pneumococcal disease (IPD) [2] . Colonisation with subsequent carriage in the upper airways is considered a "conditio sine qua non" for the development of pneumococcal respiratory tract infections and IPD [3, 4] . A critical colonisation density in the nasopharynx above which there is a high risk for pneumococcal pneumonia versus asymptomatic colonisation has been demonstrated in adults and children [5] [6] [7] .

Spn encodes and produces different quorum sensing (QS) systems including the com QS system. Recent studies showed that Com regulates colonisation and biofilms production on human lung and pharyngeal cells [8] . Com, encoded by the locus comABCD, regulate expression of genes which are involved in competence-induced bacterial killing and fratricide, production of lantibiotics (Phr peptide QS), and increased virulence due to increased production of capsular polysaccharides [9] .

To study pneumococcal-human interaction, animal models and immortalised cell culture models are generally utilised. The latter frequently consists of cancer cell lines, which represent an "artificial" environment lacking natural tissue diversity, differentiation and functionality (e.g. mucus production) of involved specialised airway cells and the 3D structure of the human lung epithelium, including the physiological air-liquid interface (ALI) and formation of tight-junctions (TJs) [10, 11] .

An ex vivo tissue model of primary human airway epithelial cells (HAE) with an air-liquid interface (ALI) was recently developed in our institution to study coronavirus pathogenesis [16] .

This model includes a pseudo-stratified HAE cell layer containing basal, secretory, columnar, and ciliated cell populations. Thus, the ex vivo system recapitulates essential aspects of human airway epithelia, namely (i) presence of well-defined human airway epithelial cell types, which become (ii) functional and differentiated by producing mucus and showing ciliary activity, and (iii) maintains physico-biochemical barriers, such as the mucus layer, and a well-formed TJ belt with development of a trans-epithelial electrical resistance (TEER). These advantageous physiological characteristics and absence of an ex vivo model of human lung infection motivated us to adopt this ex vivo HAE-ALI model. We therefore infected differentiated human lung cells with a reference Spn strain TIGR4 (wildtype) or its isogenic Δ comC mutant and comprehensively compared colonisation, invasion, translocation, cytotoxicity, and human lung epithelial cell response. insert covered by a membrane with 1 µm pores until formation of a confluent differentiated epithelial monolayer with beating cilia and mucus production at the ALI apical side. Cells were counted to 500'000 per insert by flow cytometry (BD FACSCANTO II, Becton Dickinson, United States). This process was monitored by TEER measurement until a stable mean resistance was reached.

All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

Spn strains were inoculated in a 200 µl suspension of balanced salt solution (BSS) on the apical side at different multiplicity of infection (MOI). ALI medium was added to the basolateral side. Pneumococci colonizing the apical compartment or invasive Spn in the basolateral compartment were counted by dilution and plating onto blood agar plates (measured as cfu/ml).

Inactivated TIGR4 or latex particles served as negative controls.

Primary HAE cells differentiate ex-vivo upon exposure to air To model pneumococcal colonisation and invasive disease, cells from five different donors were cultured (Fig. 1) . Integrity, and differentiation upon exposure to air [i.e., beating cilia (supplemental Movie 1-3), and mucus production] was observed by traditional light microscopy ( Fig. 1) . Confocal imaging showed ciliated structures and formation of a well-defined TJ complex, as evidenced by the presence of zonulin-1 (Fig. 2 , ZO-1 in control cells). Ultrastructure of the TJ was revealed by transmission electron microscopy (supplemental Fig. 2A -C). We confirmed by flow cytometry that HAE cells were mostly (~80%) epithelial cells [CD326+/CD31-, (supplemental 

To resemble natural lung infection where nutrients are acquired by Spn from human cells, i.e., instead from the cell culture medium, BSS was added to the apical side of differentiated HAE cells All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted December 17, 2021. ; https://doi.org/10.1101/2021.12.16.21267943 doi: medRxiv preprint before infecting the cells with wtSpn or the Δ comC mutant. Colonising Spn at the apical side reached the highest density (~10 10 cfu/ml) within 6 h of incubation after which there was no further increase ( Fig. 3A , B, C). In contrast, low Spn frequency was cultured from the basolateral side (translocation, "invasive disease") at this time point at any of the utilised inocula (Fig. 3D ).

Three-dimensional (3D) reconstruction of confocal z-stacks and analysis of orthogonal zy planes demonstrated Spn on the apical side of differentiated HAE cells (i.e., above the TJ staining) ( Fig. 2A, arrow) . Spn formed microcolonies particularly colocalising with cilia ( Fig. 2A, 2B , arrow). preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (Fig. 5A ). For both wtSpn and Δ comC, LDH levels significantly increased with longer incubation (Fig. 5A) . Higher LDH levels were observed with Δ comC mutant compared to wtSpn for all inoculation doses (Fig. 5B) . When there was no translocation, LDH levels did not differ between both strains until 19 h but were higher with Δ comC after 30 h (Fig. 5C ). In contrast, with translocation, higher LDH levels were seen with Δ comC compared to wtSpn at 19 h and 30 h (Fig. 5D) . Surprisingly, among wtSpn, inserts with translocation showed significantly lower LDH levels at 19 h and 30 h than those without translocation (Fig. 5E ). In contrast, among Δ comC, LDH levels were similar regardless of translocation (Fig. 5F ).

Spn also produces a homolog LDH enzyme during its metabolism [17] . We therefore quantified the amount of LDH in cultures of wtSpn grown for up to 30 h and LDH levels were below the limit of detection (15 U/L).

Δ comC mutant than wtSpn. However, cytotoxicity is likely not the mechanism leading to translocation.

Since intracellular pneumococci colocalised with ZO-1 staining, we quantified the release of ZO-1 into the supernatants using ELISA. ZO-1 levels were significantly higher in HAE cells infected with Δ comC mutant than wtSpn (p<0.0001; Fig. 6A , 6B). ZO-1 levels increased over time, All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (Fig. 6B ) but were independent of inoculum density ( Fig. 6C ) and were not linked to translocation (Fig. 6D ).

To investigate whether ZO-1 in the supernatant was released from damaged HAE cells or resulted from upregulated gene expression, ZO-1 mRNA was measured in infected HAE cells.

Lower ZO-1 transcripts were detected in HAE cells infected with Δ comC mutant than with wtSpn ( Fig. 7A ). This became apparent at 19 h and was significant at 30 h post-infection (Fig. 7B ), but without a dose-response effect (Fig. 7C ). There was a non-significant trend for downregulation of ZO-1 expression in inserts with translocation compared to those without translocation, both for wtSpn and Δ comC (Fig. 7) . Overall, these results confirm that increased ZO-1 levels in supernatant cannot be attributed to increased ZO-1 expression but rather to increased release of ZO-1 into the supernatant.

Temporal expression of specific host cytokines reflects colonisation and preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted December 17, 2021. ; https://doi.org/10.1101/2021.12.16.21267943 doi: medRxiv preprint secretion of IL-8 and epidermal growth factor was significantly increased at all inoculated densities (Fig. 8D) . In contrast, differentiated HAE cells infected with Δ comC mutant had significantly higher levels of G-CSF at 19 h post-infection and at 30 h increased levels of IL-1α, IL-1RA, IL-1β, IL-4, IL-8, IL-9 , TNF-α, and significantly decreased detection of several cytokines including IL-3, IL-7 and RANTES (Fig. 8E) . Together, our data reveal an increased cytotoxic phenotype upon inactivation of com quorum sensing signalling. Translocation into the basolateral space coincided with loss of epithelial integrity as shown by a TEER decrease. The host response to pneumococcal invasion was characterised by an increase of proinflammatory cytokines but it was altered when the com QS system was inactivated. Our model suggests that Com is associated with microcolony formation during colonisation and translocation, whereas inactivation of comC-mediated signalling correlates with epithelial disruption with increased cytotoxicity, and an increased inflammatory response.

The Com pathway is a well-studied QS system. It allows bacterial intercellular communication and enables fratricide, uptake of exogenous DNA and genetic recombination [8, 18] . comC encodes the competence stimulating peptide (CSP) that activates the QS system. Its inactivation results in vitro in a decreased production of early stages of biofilms [8, 19] .

Likewise, in the current study, inactivation of comC led to early reduced and disorganised pneumococcal colonisation along with increased cytotoxicity and upregulated cytokine response All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted December 17, 2021. ; https://doi.org/10.1101/2021.12.16.21267943 doi: medRxiv preprint but impaired transmigration. This is consistent with loss of a highly orchestrated host-pathogen interaction. Microinvasion was followed by an inflammatory response which was postulated as a mechanism for subsequent pneumococcal clearance [20] . Given the increased identification of hormones and molecules that block bacterial QS systems, it is likely that blocking com-QS may modulate severe cases of human pneumococcal disease. This potential mechanism is under investigation in our laboratories.

Upregulation of proinflammatory cytokines correlated with cytotoxicity. For example, levels of both IL-18 and LDH were increased in those HAE cells infected with Δ comC mutant compared to those infected with the wtSpn. The release of activated IL-18 and IL-1β is a hallmark of canonicalcaspase-1-dependent-activation of the NLRP3 inflammasome [23] . Cell death can occur by uncontrolled activation of the NLRP3 inflammasome, known as pyroptosis [24, 25] . IL-18 has proinflammatory activity both IFNγ-dependent and -independent [26] . Therefore, evidence from this model indicates that pneumococci elicit a distinct IL-18 mediated inflammatory response that can induce differentiated human lung cells to undergo pyroptosis, particularly when com-QS is inactivated.

Except for immortalised cell cultures and animal models, we are aware of only two published lung tissue models [20, 27] All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Lactate dehydrogenase (LDH) release was quantified (U/l) in supernatants of differentiated HAE cells, harvested from three different donors and inoculated with wtSpn or Δ comC mutant. All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted December 17, 2021. ; https://doi.org/10.1101/2021.12.16.21267943 doi: medRxiv preprint for different time points (B) and concentrations (C) and stratified for both strains each without translocation ("no") and with translocation ("yes") (D). All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted December 17, 2021. ; https://doi.org/10.1101/2021.12.16.21267943 doi: medRxiv preprint

Clinical and economic burden of community-acquired pneumonia among adults in Europe

A New Pneumococcal Capsule Type, 10D, is the 100th Serotype and Has a Large cps Fragment from an Oral Streptococcus

Streptococcus pneumoniae colonisation: the key to pneumococcal disease

Use of a rapid test of pneumococcal colonization density to diagnose pneumococcal pneumonia

Improvement of pneumococcal pneumonia diagnosis using quantitative real-time PCR targeting lytA in adult patients: a prospective cohort study

Nasopharyngeal Pneumococcal Colonization Density is Associated with Severe Pneumonia in Young Children in the Lao PDR

Quorum-sensing systems LuxS/autoinducer 2 and Com regulate Streptococcus pneumoniae biofilms in a bioreactor with living cultures of human respiratory cells

Autoinducer 2 Signaling via the Phosphotransferase FruA Drives Galactose Utilization by Streptococcus pneumoniae, Resulting in Hypervirulence

Human lung ex vivo infection models

Animal models of Streptococcus pneumoniae disease

Experimental human pneumococcal carriage

Psychosocial aspects of Mexican-American, white, and black teenage pregnancy

Experimental Human Pneumococcal Carriage Augments IL-17A-dependent T-cell Defence of the Lung

Experimental Human Challenge Defines Distinct Pneumococcal Kinetic Profiles and Mucosal Responses between Colonized and Non-Colonized Adults

Lactate dehydrogenase is the key enzyme for pneumococcal pyruvate metabolism and pneumococcal survival in blood

Pneumococcal Competence Coordination Relies on a Cell-Contact Sensing Mechanism

The impact of the competence quorum sensing system on Streptococcus pneumoniae biofilms varies depending on the experimental model

Microinvasion by Streptococcus pneumoniae induces epithelial innate immunity during colonisation at the human mucosal surface

Regulation of Staphylococcus aureus

The Vibrio cholerae MARTX toxin silences the inflammatory response to cytoskeletal damage before inducing actin cytoskeleton collapse

The Role of NLRP3 Inflammasome in Pneumococcal Infections

Activation mechanisms of inflammasomes by bacterial toxins

Inflammasome activation and evasion by bacterial pathogens

Interleukin-18, more than a Th1 cytokine

Streptococcus pneumoniae-induced regulation of cyclooxygenase-2 in human lung tissue

We thank Sabine Güsewell, PhD sincerely for the great expert statistical advice. All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this this version posted December 17, 2021. ;