Anaerobic microbiota promote pathogen association with the airway epithelium

TL;DR

This study shows that anaerobic bacteria in the nasal passages help a harmful bacterium, Pseudomonas aeruginosa, stick to airway cells by breaking down protective mucus.

Contribution

The study demonstrates that anaerobic microbiota enhance P. aeruginosa colonization by degrading mucin glycoproteins.

Findings

Anaerobe exposure increased epithelial inflammation and degraded mucin glycoproteins.

Anaerobe pre-treatment significantly enhanced P. aeruginosa attachment to epithelial cells.

Mucins from anaerobe-treated cells promoted more P. aeruginosa attachment in vitro.

Abstract

Introduction. Chronic rhinosinusitis (CRS) is a prevalent condition characterized by mucus stasis, persistent inflammation and infection of the paranasal sinuses. CRS often involves infection by the bacterium Pseudomonas aeruginosa, especially in individuals with cystic fibrosis or a history of antibiotic use. While P. aeruginosa is a well-established opportunistic pathogen that deploys a diverse array of virulence factors to drive airway infections, its persistence in the airway mucosa is also likely influenced by its local microbial ecology. For instance, anaerobic bacterial genera, such as Streptococcus, Veillonella and Prevotella, are also commonly found in CRS and may contribute to pathogen establishment.

Hypothesis. Although anaerobes are common members of the CRS microbiota, their role in promoting P. aeruginosa association with the airway epithelium remains poorly defined. We hypothesized that anaerobes facilitate P. aeruginosa attachment by degrading mucin glycoproteins that decorate the epithelial surface.

Aim. To determine whether CRS-associated anaerobic microbiota enhance P. aeruginosa colonization of the airways through mucin modification.

Methodology. Using a novel dual oxic-anoxic culture platform, Calu-3 epithelial cells were co-cultured with a CRS-derived anaerobic microbial community. Inflammatory gene expression, mucin integrity and subsequent P. aeruginosa epithelial association were assessed. Additionally, mucins isolated from anaerobe-treated cells were evaluated for their ability to promote P. aeruginosa attachment in vitro.

Results. Anaerobe exposure increased epithelial inflammatory marker gene expression and led to degradation of mucin glycoproteins. Anaerobe pre-treatment significantly enhanced P. aeruginosa association with the epithelial surface. Moreover, mucins isolated from anaerobe-treated cells promoted greater pathogen attachment in vitro compared to intact mucins.

Conclusion. CRS-associated anaerobic microbiota can remodel the sinonasal microenvironment in ways that enhance P. aeruginosa epithelial association. These findings highlight the importance of polymicrobial interactions in CRS pathogenesis and suggest that targeting anaerobe-mediated mucin degradation may represent a novel therapeutic strategy for chronic airway disease.

Figures

Anaerobic microbiota colonize the apical surface of Calu-3 cells and induce a pro-inflammatory response. (a) Taxonomic composition of an anaerobic bacterial consortium (ABC) derived from the upper airways. (b) SEM micrograph of Calu-3 cells after CRS challenge. (c) Bacterial recovery from Calu-3 cells after 24 h by washing with PBS, Triton X-100 and plating on Prevotella selective agar (BBA). (d) Anaerobe (ABC) challenge did not result in Calu-3 cytotoxicity relative to unchallenged O2-grown or DOAC-grown cells. (e) MA plot representation of Calu-3 gene expression under ANLI after ABC challenge relative to an untreated ANLI control. (f) Log10-normalized gene counts from five or six independent biological replicates. Data in panels (c) and (d) represent the mean and standard deviation of three biological and technical replicates (n=3) and were compared using an ordinary one-way ANOVA with multiple comparisons. Data in panels (e) and (f) represent six biological replicates per condition (n=6) and were compared using a Wald test, Benjamini–Hochberg adjusted (*P<0.001, P<0.01, *P<0.05).

Anaerobic microbiota remodel secreted mucins at the epithelial surface. (a) Representative (n=3) FPLC traces of MUC5AC mucins purified from Calu-3 cells grown at ANLI (untreated) and after treatment with an anaerobic bacterial community (ABC, treated). (b) Area under curve (AUC) for both peak 1 (high-molecular-weight mucins) and peak 2 (low-molecular-weight mucin). Data shown were derived from three independent experiments (n=3) and were compared using a unpaired t-test with Welch’s correction.

Anaerobic microbiota condition epithelial mucus to increase early pathogen association. (a) Schematic of experimental design and predicted outcomes. (b) Calu-3 pre-treatment with an anaerobic bacterial consortium (ABC) increases host cell-associated P. aeruginosa c.f.u. after 2 h challenge. (c) P. aeruginosa adhesion to microtiter plates coated with mucin (MUC5AC), ABC-treated mucin and neutrophil elastase (NE)-treated mucin relative to an uncoated control (PBS). (d) P. aeruginosa Calu-3-associated c.f.u. after pre-treatment with individual anaerobes (S.g., S. gordonii; S.p., S. parasanguinis; V.p., V. parvula; P.o., Prevotella oris; P.m., Prevotella melaninogenica). Data shown in panels (b)–(d) were derived from three independent experiments with three biological replicates (n=9). Data in (b) were compared using an unpaired t-test. Data in (c) were compared using an ordinary one-way ANOVA with multiple comparisons. Pairwise comparisons in panel (d) were compared using an unpaired t-test with Welch’s correction.

Full text

Data Summary

All code, data and raw sequence files generated in this study are available at https://github.com/Hunter-Lab-UMN/Moore_PJ_2020 and the National Center for Biotechnology Information (NCBI) Sequence Read Archive under Bioproject PRJNA902376, accession numbers SAMN31758996–SAMN31759011.

Introduction

Chronic rhinosinusitis (CRS) is a widespread and debilitating condition marked by persistent infection and inflammation of the paranasal sinuses [13]. Among bacteria linked to CRS pathology, Pseudomonas aeruginosa is the second most common isolated pathogen in CRS [4] and is frequently associated with severe and refractory cases [5], particularly in people with cystic fibrosis or prior antibiotic exposure [610]. While P. aeruginosa is a well-established opportunistic pathogen, its ability to successfully colonize the sinonasal environment is likely shaped by complex interactions with co-colonizing microbiota.

Strict and facultative anaerobic bacterial genera (Streptococcus, Veillonella, Prevotella, Fusobacterium and Porphyromonas), which are also both prevalent and abundant among sinonasal microbiota [911], have increasingly been recognized as potential facilitators of P. aeruginosa persistence and pathogenicity [1215]. Previous studies suggest that anaerobes and their metabolites enhance P. aeruginosa biofilm formation [131617], expand nutrient accessibility [121819], alter antimicrobial susceptibility of airway pathogens [2023] and modulate immune responses in ways that may favour P. aeruginosa persistence [24]. Although these findings have primarily been observed in lower airway diseases like cystic fibrosis, it remains unclear whether anaerobic bacteria promote P. aeruginosa colonization of the sinuses and, if so, what specific molecular mechanisms are involved.

A notable challenge in studying the anaerobe–pathogen–host dynamic is the lack of laboratory models that allow co-culture of oxygen-dependent epithelial cells and oxygen-sensitive anaerobes. To overcome this, we recently developed a dual oxic–anoxic culture platform (DOAC), in which polarized epithelial monolayers are cultured in an anoxic workstation while oxygen is supplied to the basolateral compartment (Fig. S1, available in the online Supplementary Material) [25]. This setup creates a near-anoxic microenvironment on the apical surface, permitting co-culture of airway epithelial cells with strict and facultative anaerobes and the tractable evaluation of their interactions over time. Although Calu-3 cells are derived from the lower airway, differentiation at air–liquid interface yields polarized, mucus-producing epithelia with abundant secreted gel-forming mucins, enabling controlled testing of anaerobe–mucin–pathogen interactions at a mucus-decorated epithelial surface. Accordingly, this reductionist model is used to interrogate mechanisms of mucus layer remodelling and early pathogen association, rather than to recapitulate sinonasal-specific epithelial differentiation or CRS tissue remodelling.

In this study, we use DOAC to evaluate the role of CRS-associated anaerobes (e.g. Streptococcus, Veillonella and Prevotella spp.) in promoting P. aeruginosa early association with the mucus-conditioned epithelial surface. Specifically, we test the hypothesis that anaerobes condition a mucosal microenvironment conducive to P. aeruginosa colonization through the modification and degradation of mucin glycoproteins. Using a CRS-derived anaerobic microbial community to challenge mucus-overproducing Calu-3 epithelial cells [2628], we observed limited cytotoxicity but enhanced inflammatory gene expression, modification of low-molecular-weight mucins and enhanced pathogen association with variations depending on the specific anaerobe. Understanding the molecular and ecological interactions that enable P. aeruginosa to establish itself and thrive in the mucus-rich, oxygen-limited upper airways could provide valuable insights into CRS pathogenesis and reveal new therapeutic strategies to disrupt bacterial synergy.

Methods

Epithelial cell culture

Calu-3 cells were cultured in Minimal Essential Medium (MEM) (Corning, USA) supplemented with 10% foetal bovine serum (Gene) and antibiotics (100 U ml^−1^ penicillin and 100 µg ml^−1^ streptomycin)(Gibco) at 37 °C in a 5% CO_2_ incubator. Once the cells reached 80% confluency, 1×10^5^ cells were seeded onto 6.5 mm culture inserts (0.4 µm pore) (Corning). After ~5 days, the apical medium was removed to establish an air–liquid interface (ALI). Cells were then maintained for an additional 21–28 days to allow differentiation and mucus accumulation.

To assemble the cell cultures in a gas-permeable multi-well plate manifold (DOAC) (Fig. S1), fully polarized Calu-3 cells were placed into a custom 3D-printed gasket fitted within a 24-well gas-permeable plate (CoyLabs, Grass Lake, MI) containing 800 µl of MEM per well. Sterile mineral oil (400 µl) was added to unused wells to prevent unwanted gas exchange. Once the assembled system was transferred into an anaerobic chamber (90% N_2_/5% H_2_/5% CO_2_), a mixed gas flow (21% O_2_/5% CO_2_/74% N_2_) was supplied to the plate base to oxygenate the basolateral compartment. A schematic of this setup is provided in Fig. S1.

Fast protein liquid chromatography

Secreted mucins were collected from Calu-3 cells as previously described [29]. Briefly, cells grown on Transwell inserts were solubilized in a reduction buffer containing 6M guanidine hydrochloride, 0.1M Tris-HCl and 5 mM EDTA (pH 8). To minimize mucin degradation, 10 mM DTT and a cOmplete Mini protease inhibitor tablet (Roche) were added to 400 ml of reduction buffer before solubilization. Cell suspensions were gently agitated by pipetting to dislodge biomass, and suspensions from six Transwell inserts per plate were pooled into a single aliquot. Cells were rinsed with reduction buffer to remove residual mucin. This mixture was incubated at 37 °C for 5 h, followed by the addition of 25 mM iodoacetamide and overnight incubation at room temperature. Mucins were then dialysed (1,000 kDa MWCO) against 1l of 4M GuHCl buffer containing 2.25 mM NaH_2_PO_4_-H_2_O and 76.8 mM Na_2_HPO_4_ for 36 h, with buffer exchanges every 12 h.

To evaluate the integrity of high-molecular-weight mucins, size-exclusion chromatography was performed using an Äkta Pure FPLC system (GE Healthcare BioSciences, Marlborough, MA) at 4 °C. A 500 µl aliquot of purified mucin was manually injected and subjected to an isocratic run at a 0.4 ml min^−1^ flow rate for 1.5 column volumes using a 15-ml 10/200 Tricorn column packed with Sepharose 4B-CL beads. The mobile phase consisted of 150 mM NaCl in 50 mM phosphate buffer (pH 7.2). Data were collected using Unicorn software (GE Healthcare Biosciences).

Bacterial strains and culture conditions

P. aeruginosa PA14 was routinely cultured on Luria–Bertani (LB) medium. The anaerobic strains Prevotella melaninogenica ATCC 25845, Streptococcus parasanguinis ATCC 15912 and Veillonella parvula ATCC 10790 were obtained from Microbiologics (St. Cloud, MN). Streptococcus gordonii was obtained from M.C. Herzberg (University of Minnesota) and Prevotella oris 12252T was purchased from the Japan Collection of Microorganisms. All anaerobes were maintained on brain–heart infusion (BHI) medium supplemented with 0.25 g l^−1^ hemin, 0.025 g l^−1^ vitamin K and 5% (vol/vol) laked sheep’s blood (BHI-HKB) under anaerobic conditions. Additionally, a mucin-enriched anaerobic bacterial community (ABC) isolated from an individual with CRS was enriched and cultured in a minimal mucin medium previously described [1230].

Quantification of P. aeruginosa association with epithelial cultures

Following anaerobe pre-treatment, Calu-3 ALI cultures were apically challenged with ~5×10^7^ c.f.u. of P. aeruginosa PA14 for 2 h under DOAC conditions. After infection*,* cultures were washed with sterile PBS to remove non-associated bacteria and then treated with 0.25% Triton-X 100 to release bacteria remaining associated with the epithelium. Recovered bacteria were enumerated by plating serial dilutions on LB agar. Because bacteria were recovered after washing and detergent permeabilization, c.f.u. counts represent the total cell-associated bacterial population at the sampling time point and include tightly adherent bacteria and any bacteria not removed by washing. This assay was not designed to distinguish surface attachment from internalization or early microcolony formation.

RNA sequencing

The transcriptomic response of Calu-3 cells to anaerobe challenge was analysed using RNAseq. Calu-3 cells were cultured at an air–liquid interface and harvested after 24 h of incubation under DOAC conditions with or without bacterial challenge. Normoxic (unchallenged) controls were maintained under standard incubator conditions. At the conclusion of each experiment, RNAlater (Invitrogen) was added to both the apical and basolateral compartments. RNA was extracted from five or six Transwells using the RNeasy Micro Plus kit (QIAGEN) following the manufacturer’s instructions. DNase treatment was performed using the Zymo RNA Clean and Concentrator kit. RNA quality (RIN >9.7) was assessed using an Agilent Bioanalyzer, and RNA quantity was measured using RiboGreen. cDNA libraries were prepared using the SMARTer Universal Low Input RNA Kit (Takara Bio) and sequenced on an Illumina NovaSeq 6000 platform at the University of Minnesota Genomics Center.

For analysis, the Ensembl GTF annotation file was filtered to exclude non-protein-coding features. Fastq files were subsampled to a maximum of 100,000 reads per sample, and data quality was assessed with FastQC. Raw reads were aligned to the Homo sapiens reference genome (GRCh38) with Ensembl release 98 annotations. Gene counts were generated using the ‘featureCounts’ function of the RSubread package [31]. Differential expression analysis was performed using DESeq2 (v.1.28.1), where size factors were estimated, gene-wise dispersions were computed, shrinkage was applied (type=‘ashr’) and Wald hypothesis testing was conducted [3233]. Genes with a log_2_ fold-change >1 and a Benjamini–Hochberg adjusted P-value <0.001 were considered significant. All code, data and raw sequence files are available at https://github.com/Hunter-Lab-UN/Moore_PJ_2020 and the National Center for Biotechnology Information (NCBI) Sequence Read Archive under accession number PRJNA902376.

Scanning electron microscopy

Untreated, anaerobe-challenged and P. aeruginosa-infected cell cultures were washed three times in 0.2M sodium cacodylate buffer before fixation in a primary fixative solution (0.15 M sodium cacodylate buffer, pH 7.4, 2% paraformaldehyde, 2% glutaraldehyde, 4% sucrose and 0.15% alcian blue 8 GX) for 22 h. Transwell membranes were washed three more times and treated with secondary fixative (1% osmium tetroxide, 1.5% potassium ferrocyanide, 0.135M sodium cacodylate and pH 7.4) for 90 min. After three additional washes, cells were dehydrated in a graded ethanol series (25%, 50%, 75%, 85%, 2×95% and 2×100%) for 10 min per step, followed by CO_2_-based critical point drying. Transwell membranes were then mounted on scanning electron microscopy (SEM) specimen stubs using carbon conductive adhesive tape and sputter-coated with ~5 nm iridium using the Leica ACE 600 magnetron-based system. Imaging was performed on a Hitachi S-4700 field emission SEM at an operating voltage of 2 kV. Images were false coloured using Adobe Photoshop CS6.

Microtiter plate binding assay

The adhesion of P. aeruginosa to mucus was tested using an established microtiter plate-based assay [34]. 96-well MaxiSorp microtiter plates (Nunc) were coated with 40 µg ml^−1^ of mucins (MUC5AC) derived from untreated and ABC-treated Calu-3 cells. As a control, MUC5AC treated with neutrophil elastase (1 µg ml^−1^) was also used. After coating, the plates were incubated for 24 h at 37 °C. Mucin-coated wells were then washed three times with sterile PBS to remove any residual unbound mucin. Next, 5×10^7^ c.f.u. of P. aeruginosa PA14 were added to mucin-coated wells and incubated for an additional 2 h at 37 °C. Wells were washed 10 times with PBS to remove any unbound bacteria. Bound PA14 was desorbed by treating the wells with 200 µl of 0.25% Triton X-100 for 15 min at room temperature. Bacteria bound to each well were enumerated by plating serial dilutions on LB agar. All assays were performed using three biological replicates and three technical replicates.

Results

Anaerobic airway microbiota promote an inflammatory host response

The lack of tractable epithelial cell culture systems compatible with hypoxic or anoxic growth has limited our understanding of host–anaerobe interactions. Prior work has shown that culture supernatants of anaerobic bacteria elicit pro-inflammatory cytokine expression in vitro through mixed-acid fermentation and production of SCFAs [183536]. However, it is not yet known how the host responds to the physical presence of anaerobes at the airway epithelial interface. To address this knowledge gap, we used the DOAC model to assess the response of immortalized Calu-3 airway epithelial cells to co-culture with CRS-associated anaerobic microbiota.

We first utilized a defined ABC isolated from the upper airways of an individual with chronic sinusitis. This representative community was derived through anaerobic enrichment culturing of surgically collected sinus mucus and was selected for its dominant bacterial taxa (Veillonella, Prevotella and Streptococcus), which are associated with chronic sinus disease (Fig. 1a). These genera are also known to degrade mucin glycoproteins that decorate the surface of Calu-3 cells, support pathogen growth through cross-feeding and enhance P. aeruginosa pathogenicity both in vitro and in vivo [12141530]. After 3 h of equilibration in the DOAC system (i.e. oxygenated basolateral compartment and anoxic apical surface), Calu-3 cells were apically challenged with the ~8×10^6^ c.f.u. of the anaerobic community and incubated for an additional 24 h. Colonization was confirmed using scanning electron microscopy, which revealed bacterial cells at the epithelial interface (Fig. 1b). Importantly, anaerobes (4×10^6^ c.f.u.) were recovered after 24 h by washing with PBS and plating on BHI agar (Fig. 1c). Washing with Triton X-100 resulted in a 0.7-log increase in recovery (1.6×10^7^ c.f.u.), indicating robust association with the epithelial layer, consistent with bacteria that are tightly adherent and/or not removed by PBS washing of host cells (i.e. bacterial cells were not removed by PBS washing alone). Recovery of ~7×10^5^ c.f.u. on a *Prevotella-*selective medium (Brucella Blood Agar, BBA) under anaerobic conditions confirmed that apical oxygen concentrations were sufficiently low to support strict anaerobe growth, consistent with our previous work showing anaerobe proliferation and mixed-acid fermentation under DOAC conditions [25]. Despite this bacterial growth on the apical surface of the epithelial cells, cytotoxicity was not induced by anaerobe challenge after 24 h (Fig. 1d).

*Anaerobic microbiota colonize the apical surface of Calu-3 cells and induce a pro-inflammatory response. (a) Taxonomic composition of an anaerobic bacterial consortium (ABC) derived from the upper airways. (b) SEM micrograph of Calu-3 cells after CRS challenge. (c) Bacterial recovery from Calu-3 cells after 24 h by washing with PBS, Triton X-100 and plating on Prevotella selective agar (BBA). (d) Anaerobe (ABC) challenge did not result in Calu-3 cytotoxicity relative to unchallenged O2-grown or DOAC-grown cells. (e) MA plot representation of Calu-3 gene expression under ANLI after ABC challenge relative to an untreated ANLI control. (f) Log10-normalized gene counts from five or six independent biological replicates. Data in panels (c) and (d) represent the mean and standard deviation of three biological and technical replicates (n=3) and were compared using an ordinary one-way ANOVA with multiple comparisons. Data in panels (e) and (f) represent six biological replicates per condition (n=6) and were compared using a Wald test, Benjamini–Hochberg adjusted (***P<0.001, **P<0.01, P<0.05).

We then used RNAseq to profile the transcriptional response of Calu-3 cells to the anaerobe community (Fig. 1e and Data S1). Contrary to our expectations, only five genes showed differential expression relative to untreated cell cultures, all of which were upregulated (log2 fold change ≥1, adjusted P-value<0.001). These genes were all markers of inflammation and included ICAM1, TNFAIP2 (which is regulated by TNFα in response to bacterial challenge) [37], chemokines CXCL1 and CXCL5 (which act as neutrophil chemoattractants and are primarily expressed during acute inflammation) [3839] and SOD2 (superoxide dismutase 2, which is expressed in response to lipopolysaccharide and protects against apoptosis caused by inflammatory cytokines) [40]. Additional inflammatory markers, such as PI3 (elafin, an elastase inhibitor that primes innate immune responses in the lung) [41], CXCL3, C3 (complement), CYP24A1 (cytochrome p450 family 24 subfamily A member 1), OAS1 (oligoadenylate synthetase) and SDC4 (syndecan 4), were also differentially expressed, but did not reach statistical significance (Fig. 1f).

Anaerobic microbiota remodel the mucus layer via mucin degradation

Previous work by us and others demonstrated the functional capacity of anaerobic microbiota to degrade airway mucins and support the growth of canonical pathogens via nutrient cross-feeding [123042]. Thus, in support of downstream pathogen colonization experiments, we used FPLC to determine whether anaerobe challenge altered Calu-3 mucin integrity relative to unchallenged cells. To do so, we collected and purified mucin from the apical side of the Transwells as previously described [29] and used size-exclusion chromatography to assay their integrity. As expected, chromatograms revealed two characteristic peaks: (i) high-molecular-weight mucins which ran in the void volume of the column and (ii) a broader inclusion volume peak representative of lower-molecular-weight mucins [4344] (Fig. 2a). While differences in the chromatographic profile of peak 1 (high-molecular-weight mucins) were negligible between culture conditions (P=0.38), peak 2 area was significantly reduced (P=0.007) following anaerobe challenge (Fig. 2b), reflecting degradation of low-molecular-weight mucin glycoproteins. These data suggest that in addition to eliciting an inflammatory host response, anaerobic colonization alters the physicochemical properties of the mucin-decorated epithelial surface by preferentially reducing the low-molecular-weight fraction.

Anaerobic microbiota remodel secreted mucins at the epithelial surface. (a) Representative (n=3) FPLC traces of MUC5AC mucins purified from Calu-3 cells grown at ANLI (untreated) and after treatment with an anaerobic bacterial community (ABC, treated). (b) Area under curve (AUC) for both peak 1 (high-molecular-weight mucins) and peak 2 (low-molecular-weight mucin). Data shown were derived from three independent experiments (n=3) and were compared using a unpaired t-test with Welch’s correction.

Anaerobes increase early P. aeruginosa association with the epithelial surface

Recent studies have shown that viral challenge of the respiratory epithelium enhances P. aeruginosa colonization via interferon-mediated effects [45]. Other work has demonstrated that Streptococcus mitis compromises the protective role of the mucus barrier by hydrolysing mucin glycans [46]. Given that the anaerobe consortium in our model both triggered an inflammatory response and altered mucin integrity, we hypothesized that, in addition to providing nutrients for pathogen growth through cross-feeding [12], anaerobic microbiota enhance P. aeruginosa association with the airway epithelium (illustrated in Fig. 3a).

Anaerobic microbiota condition epithelial mucus to increase early pathogen association. (a) Schematic of experimental design and predicted outcomes. (b) Calu-3 pre-treatment with an anaerobic bacterial consortium (ABC) increases host cell-associated P. aeruginosa c.f.u. after 2 h challenge. (c) P. aeruginosa adhesion to microtiter plates coated with mucin (MUC5AC), ABC-treated mucin and neutrophil elastase (NE)-treated mucin relative to an uncoated control (PBS). (d) P. aeruginosa Calu-3-associated c.f.u. after pre-treatment with individual anaerobes (S.g., S. gordonii; S.p., S. parasanguinis; V.p., V. parvula; P.o., Prevotella oris; P.m., Prevotella melaninogenica). Data shown in panels (b)–(d) were derived from three independent experiments with three biological replicates (n=9). Data in (b) were compared using an unpaired t-test. Data in (c) were compared using an ordinary one-way ANOVA with multiple comparisons. Pairwise comparisons in panel (d) were compared using an unpaired t-test with Welch’s correction.

To test this hypothesis, Calu-3 cells were first treated with the anaerobic consortium (shown in Fig. 1a) for 24 h and then washed to remove spent medium and unbound cells. The cells were subsequently infected with ~5×10^7^ c.f.u. of P. aeruginosa PA14 for 2 h. Removing apical culture media and limiting the incubation time ensured that any difference in Calu-3-associated c.f.u. between the anaerobe-treated cells and untreated (i.e. no anaerobe) control reflects early association with the epithelial surface under conditions designed to minimize replication. P. aeruginosa cell-associated c.f.u. were quantified by washing and permeabilizing the Calu-3 cells, followed by plate enumeration. Because the endpoint is c.f.u. recovered from washed cultures following detergent permeabilization, this assay reports the total cell-associated bacterial population (i.e. surface-adherent bacteria plus any bacteria that may be internalized) and does not distinguish attachment from invasion of early microcolony formation. As predicted, anaerobe pre-treatment resulted in an ~67% increase in P. aeruginosa c.f.u. (1.1×10^7^) relative to untreated controls (P=0.0003, Fig. 3b).

To further confirm that enhanced pathogen association was due, at least in part, to mucin degradation, mucins isolated from Calu-3 cells treated with anaerobes (and untreated controls) were used to coat the surface of a microtiter plate, followed by the addition of P. aeruginosa PA14. Consistent with recent work showing that mucins disperse P. aeruginosa biofilm growth [47], mucin coating led to a 1.5 log-reduction in bacterial attachment compared to uncoated plates (PBS alone), as expected. In contrast, anaerobe-degraded mucins showed a significant increase (P=0.0007) in P. aeruginosa binding to the microtiter plate compared to untreated mucins (Fig. 3c). To ensure that our mucin purification process (e.g. using guanidine hydrochloride) did not affect P. aeruginosa viability, plates were also coated with mucins degraded with human neutrophil elastase (NE) and isolated using the same procedure. NE-treated mucins led to similar PA14 attachment to PBS controls, confirming bacterial viability and showing that anaerobic microbiota can enhance pathogen colonization of a mucin-coated surface.

Finally, to assess the contributions of individual anaerobes to P. aeruginosa colonization, we challenged Calu-3 cells with representative isolates of the three most abundant genera in the anaerobic consortium (Streptococcus, Veillonella and Prevotella) prior to P. aeruginosa colonization (Fig. 3d). Contrary to a recent study [46], we found that Streptococcus species (S. gordonii and * S. parasanguinis*) had little effect on PA14 colonization, despite their known mucin degradation capacity [48]. Similarly, V. parvula resulted in no significant differences between treatment conditions. In contrast, challenge with both Prevotella melaninogenica and Prevotella oris, two species commonly associated with inflammatory airway disease, led to significantly increased PA14 recovery from Calu-3 cells compared to unconditioned controls (*P=*0.0003 and *P=*0.0004, respectively). These data demonstrate that while anaerobic microbiota of the upper airways facilitate enhanced early P. aeruginosa association with epithelial cultures, this effect is species-specific.

Discussion

Anaerobic bacteria have long been detected in the upper airways by culture [4951], but the advent of culture-independent sequencing has renewed interest in their role(s) in chronic sinusitis. While in vitro data support potential pathogenic mechanisms, their relevance remains unclear due to a lack of suitable anaerobe-host interaction models compatible with their conflicting oxygen demands. To address this, we developed an in vitro platform for extended co-culture of polarized airway epithelial cells with anaerobic microbiota [25]. Using this model, here we show that anaerobes commonly associated with the oral cavity – Streptococcus, Prevotella and Veillonella spp. – may help establish a mucosal inflammatory environment that promotes P. aeruginosa growth [4552]. Additionally, we demonstrate that these anaerobes enhance pathogen association with the airway epithelial surface by degrading mucin glycans. Importantly, while our data support remodelling of secreted mucins and increased pathogen association with the epithelium, we did not directly assess tight junction integrity (e.g. transepithelial electrical resistance, paracellular permeability and junctional protein localization). Accordingly, we use the term ‘interface remodelling’ to denote changes in the mucus layer and its surface properties, rather than disruption of epithelial barrier function.

Previously, we reported that anaerobes stimulate pathogen growth through mucin-based cross-feeding [1230]. Since P. aeruginosa cannot efficiently catabolize mucins alone [42], it relies on anaerobe-mediated breakdown and fermentation of mucin polypeptides and O-linked glycans for access to bioavailable substrates. Though not directly tested here, it is plausible that pre-colonization with anaerobes liberates mucin-derived metabolites, supporting pathogen persistence at the epithelial interface. While selective degradation of low-molecular-weight mucins remains unclear, our FPLC data confirm that anaerobic colonization structurally modifies the apical surface. Given that mucin degradation can diminish the protective mucus barrier that normally restricts bacterial contact with the epithelium [46], we propose that beyond the canonical mucus stagnation that accompanies CRS, anaerobic microbiota contribute to disease morbidity through a multifactorial process involving inflammation, cross-feeding and anaerobe-driven mucin remodelling.

Focusing on an early time point post-P. aeruginosa challenge (2 h), we evaluated anaerobic mucin degradation and its role in reshaping the epithelial interface to enhance pathogen attachment, as seen previously with S. mitis and Neisseria meningitidis [46]. As expected, anaerobe degradation of apical mucus significantly increased host cell-associated P. aeruginosa, which has important clinical implications. For example, epithelial colonization accelerates P. aeruginosa biofilm maturation, increases extracellular polysaccharide production and induces quorum-sensing and other transcriptional changes [53]. Additionally, P. aeruginosa grown on bronchial epithelial cells is far more resistant to antibiotics than when cultured on abiotic surfaces [53], consistent with its increased tolerance in vivo. We posit that anaerobe-mediated colonization further potentiates these phenotypes. Future studies will be needed to evaluate P. aeruginosa–host interactions over longer time periods to further understand differences in pathogen physiology and the inflammatory host response in the presence of anaerobic microbiota.

Limitations of this work include the use of an immortalized lower-airway epithelial model rather than primary sinonasal cultures. Primary cells and CRS mucus exhibit disease- and region-specific differences in cell type and differentiation state, inflammatory tone and mucin composition that may modulate the effects reported here. Nonetheless, the DOAC platform provides a tractable model system to test the concept that anaerobic consortia can enzymatically remodel airway mucins in a manner that increases early pathogen association with the epithelium. Future work with primary cells or patient-derived mucus will be important to determine whether these effects are conserved in more CRS-relevant contexts and more broadly across diverse epithelial surfaces.

In conclusion, our findings highlight the potential role of anaerobic microbiota in shaping the sinonasal environment to favour P. aeruginosa colonization. By degrading mucin glycans and altering the epithelial interface, anaerobes may facilitate pathogen attachment and persistence, contributing to the onset and progression of chronic sinusitis and its recalcitrance to antibiotic therapy. Given the well-documented challenges of treating P. aeruginosa infections [54], particularly in biofilm-associated states, understanding the interplay between anaerobes, canonical airway pathogens and the host will be critical. We are currently evaluating the mechanistic basis of anaerobe-mediated mucus remodelling, its consequences for pathogen association and potential therapeutic strategies to mitigate their impacts on airway disease.

Supplementary material

10.1099/jmm.0.002149Uncited Fig. S1.

10.1099/jmm.0.002149Uncited Supplementary Material 1.