Mono- and co-infections of primary porcine respiratory cells with Bordetella bronchiseptica and Streptococcus suis are not affected by the dermonecrotic toxin.

Mono- and co-infections of primary porcine respiratory cells with Bordetella bronchiseptica and Streptococcus suis are not affected by the dermonecrotic toxin - PMC Skip to main content An official website of the United States government Here's how you know Here's how you know Official websites use .gov A .gov website belongs to an official government organization in the United States. Secure .gov websites use HTTPS A lock ( Lock Locked padlock icon ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites. Search Log in Dashboard Publications Account settings Log out Search… Search NCBI Primary site navigation Search Logged in as: Dashboard Publications Account settings Log in Search PMC Full-Text Archive Search in PMC Journal List User Guide PERMALINK Copy As a library, NLM provides access to scientific literature. 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Learn more: PMC Disclaimer | PMC Copyright Notice Infect Immun . 2026 Mar 12;94(4):e00366-25. doi: 10.1128/iai.00366-25 Search in PMC Search in PubMed View in NLM Catalog Add to search Mono- and co-infections of primary porcine respiratory cells with Bordetella bronchiseptica and Streptococcus suis are not affected by the dermonecrotic toxin Désirée Schaaf Désirée Schaaf 1 Institute for Microbiology, University of Veterinary Medicine Hannover, Hanover, Germany Conceptualization, Data curation, Investigation, Methodology, Project administration, Validation, Visualization, Writing – original draft, Writing – review and editing Find articles by Désirée Schaaf 1, ✉ , Muriel Dresen Muriel Dresen 1 Institute for Microbiology, University of Veterinary Medicine Hannover, Hanover, Germany Data curation, Investigation, Methodology, Visualization, Writing – review and editing Find articles by Muriel Dresen 1 , Yenehiwot Berhanu Weldearegay Yenehiwot Berhanu Weldearegay 1 Institute for Microbiology, University of Veterinary Medicine Hannover, Hanover, Germany Data curation, Investigation, Methodology Find articles by Yenehiwot Berhanu Weldearegay 1 , Jeannine Biermann Jeannine Biermann 1 Institute for Microbiology, University of Veterinary Medicine Hannover, Hanover, Germany Data curation, Investigation Find articles by Jeannine Biermann 1 , Susan L Brockmeier Susan L Brockmeier 2 Virus and Prion Research Unit, National Animal Disease Center, ARS, USDA, Ames, Iowa, USA Resources Find articles by Susan L Brockmeier 2 , Wolfgang Baumgärtner Wolfgang Baumgärtner 3 Department of Pathology, University of Veterinary Medicine Hannover, Hanover, Germany Resources Find articles by Wolfgang Baumgärtner 3 , Michael Jarek Michael Jarek 4 Genome Analytics (GMAK), Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany Data curation, Investigation Find articles by Michael Jarek 4 , Peter Valentin-Weigand Peter Valentin-Weigand 1 Institute for Microbiology, University of Veterinary Medicine Hannover, Hanover, Germany Conceptualization, Funding acquisition, Methodology, Resources, Supervision, Writing – review and editing Find articles by Peter Valentin-Weigand 1 Editor: Denise M Monack 5 Author information Article notes Copyright and License information 1 Institute for Microbiology, University of Veterinary Medicine Hannover, Hanover, Germany 2 Virus and Prion Research Unit, National Animal Disease Center, ARS, USDA, Ames, Iowa, USA 3 Department of Pathology, University of Veterinary Medicine Hannover, Hanover, Germany 4 Genome Analytics (GMAK), Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany 5 Stanford University School of Medicine, Stanford, California, USA ✉ Address correspondence to Désirée Schaaf, [email protected] The authors declare no conflict of interest. Roles Désirée Schaaf : Conceptualization, Data curation, Investigation, Methodology, Project administration, Validation, Visualization, Writing – original draft, Writing – review and editing Muriel Dresen : Data curation, Investigation, Methodology, Visualization, Writing – review and editing Yenehiwot Berhanu Weldearegay : Data curation, Investigation, Methodology Jeannine Biermann : Data curation, Investigation Susan L Brockmeier : Resources Wolfgang Baumgärtner : Resources Michael Jarek : Data curation, Investigation Peter Valentin-Weigand : Conceptualization, Funding acquisition, Methodology, Resources, Supervision, Writing – review and editing Denise M Monack : Editor Received 2025 Jul 10; Accepted 2026 Feb 3; Collection date 2026 Apr. Copyright © 2026 Schaaf et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license . PMC Copyright notice PMCID: PMC13081715  PMID: 41817187 ABSTRACT Bordetella bronchiseptica is a gram-negative bacterium contributing to respiratory diseases in many different animal species. In the swine population, it occurs frequently and plays a role in the Porcine Respiratory Disease Complex as well as in the pathogenesis of atrophic rhinitis. The dermonecrotic toxin (DNT) is involved in the destruction of the nasal conchae, a hallmark of atrophic rhinitis, and several studies have shown the effects of DNT on osteoblastic cells. Surprisingly, only little is known about the interactions of DNT and respiratory epithelial cells. Thus, we investigated the influence of DNT on porcine respiratory epithelial cells during mono- and co-infections in vitro . For this, we infected porcine precision-cut lung slices and air-liquid interface cultures with a DNT-positive B. bronchiseptica wild-type strain and its isogenic DNT-deficient mutant strain. For co-infection experiments, a Streptococcus suis serotype 2 wild-type strain was used. We evaluated cytotoxic effects and colonization of both pathogens, as well as the pro-inflammatory cytokine response of the host cells. Remarkably, DNT neither contributed to the cytotoxic effects of B. bronchiseptica nor did it affect bacterial colonization. Regarding the cytokine response, pro-inflammatory cytokines were expressed mainly upon infection with B. bronchiseptica but hardly after infection with S. suis , whereas co-infection with both pathogens had an amplifying effect on cytokine expression after prolonged infection, independently of DNT. Concluding, we found no evidence that DNT contributes to the early stages of infection with B. bronchiseptica and S. suis in in vitro models of the porcine respiratory tract. KEYWORDS: air-liquid interface cultures, porcine precision-cut lung slices, porcine respiratory tract infection, Streptococcus suis , dermonecrotic toxin, Bordetella bronchiseptica INTRODUCTION Bordetella bronchiseptica is a small, rod-shaped, and gram-negative bacterium that is closely related to the human pathogen Bordetella pertussis , the causative agent of whooping cough. In contrast to its host-specific relative, B. bronchiseptica can cause respiratory infections of multiple etiology in many different animal species as well as in humans. In pigs, B. bronchiseptica is the causative agent of non-progressive atrophic rhinitis, which can, in the presence of toxigenic Pasteurella multocida , lead to the severe progressive form ( 1 ). Turbinate atrophy caused by B. bronchiseptica can be associated with the dermonecrotic toxin (DNT), a cytoplasmic, heat-labile toxin belonging to the group of cytotoxic necrotizing factors that is almost identical in B. bronchiseptica and B. pertussis ( 2 – 4 ). In vitro studies have shown that purified DNT induces tremendous morphological changes of the cytoskeleton due to DNT-mediated activation of the small GTP-binding protein Rho and other members of the Rho family ( 5 ). Moreover, it can inhibit the differentiation of osteoblastic cells ( 6 ), resulting in deformation of the nasal conchae as typically seen in atrophic rhinitis. However, to our knowledge, nothing is known about the effects of DNT on respiratory epithelial cells. Recently, the T-type voltage-gated calcium channels Ca v 3.1 and Ca v 3.2 were identified as DNT-binding receptors in different mouse cell lines ( 3 ), which are located on neurons, cardiac, and skeletal cells, but are also expressed by human lung epithelial cells ( 7 ). As part of the Porcine Respiratory Disease Complex (PRDC), B. bronchiseptica also contributes to pneumonia in pigs and can pave the way for infection with secondary pathogens, for example, Streptococcus suis , as we have shown in our previous study ( 8 ). S. suis is a facultative pathogenic, gram-positive bacterium colonizing the upper respiratory tract of almost all pigs. However, this pathobiont can become invasive when the respiratory epithelial barrier has been damaged by a previous infection with, for example, swine influenza virus (SIV) ( 9 , 10 ) and cause severe systemic diseases in piglets. Some studies have been published on interactions between B. bronchiseptica and S. suis, but it is yet unclear how B. bronchiseptica can predispose to infection with S. suis . Thus, in this study, we focused on (i) the role of DNT in interactions between the two pathogens and (ii) the following pro-inflammatory cytokine response of the host to co-infection with B. bronchiseptica and S. suis . We assumed that DNT can contribute to the cytotoxic effects and the colonization of B. bronchiseptica , as the latter was described in a previous in vivo study ( 11 ), consequently affecting also co-infection with S. suis . Furthermore, we expected immunomodulatory effects of a pre-infection with B. bronchiseptica as well as a synergistic pro-inflammatory response induced by both pathogens, as it has been described for other co-infections of the (porcine) respiratory tract ( 12 – 15 ). As only a little is known about the immune response during porcine respiratory tract infection with B. bronchiseptica , we set out to investigate this in our in vitro infection models—porcine respiratory epithelial cells well-differentiated under air-liquid interface conditions (ALI cultures) and the porcine precision-cut lung slice model (PCLS). Both models mimic the in vivo respiratory epithelium very closely, as they consist of ciliated and mucus-producing respiratory epithelial cells. The main advantage of ALI cultures is the formation of a barrier and the possibility to assess its integrity by resistance measurements or immunofluorescence microscopy ( 16 ). The benefit of PCLS is the preservation of the original tissue architecture and functionality, the presence of resident immune cells and other cell types, as well as the possibility to monitor the activity of ciliated cells by light microscopy ( 17 ). Thus, we made use of the advantages of both models and found that DNT is neither involved in the reduction of the ciliary activity nor in the destruction of the respiratory epithelial barrier during B. bronchiseptica mono-infection. Moreover, DNT does not contribute to colonization or to immune activation of the respiratory epithelium by B. bronchiseptica . Consequently, we found no evidence that DNT of B. bronchiseptica facilitates co-infection with S. suis . Notably, activation of pro-inflammatory cytokines was clearly induced after infection with B. bronchiseptica, but not S. suis, whereas higher levels of some cytokines (e.g., IL-1α, CXCL8) were detected after co-infection with both pathogens. MATERIALS AND METHODS Bacterial strains The wild-type strain B. bronchiseptica KM22 (WT) is a virulent phase I isolate from a swine herd with clinical atrophic rhinitis. The dnt -deficient mutant strain KM24 (Δ dnt ; in a previous study designated as “KB24”) was generated by triparental mating and insertion of a 3.7-kb Gen r /oriT cassette ( 11 ). Both strains were kindly provided by Susan L. Brockmeier (National Animal Disease Center, USA). Illumina next-generation sequencing was performed to confirm the disruption of the dnt gene in the mutant strain KM24. Raw sequence data have been deposited in the NCBI Sequence Read Archive under accession number PRJNA1214268, and the genome of KM24 is available in NCBI GenBank under accession number CP181209 . Both strains were grown on Columbia agar plates supplemented with 7% sheep blood (Oxoid, Thermo Fisher Scientific, Cat. No. PB5008A) for 48 h at 37°C under aerobic conditions as previously described ( 8 ). For infection experiments, cryo-preserved bacteria were used, and infection stocks were prepared as previously described ( 8 ), with minor modifications. Briefly, B. bronchiseptica WT and Δ dnt were grown overnight at 37°C on a horizontal shaker at 150 rounds per minute (rpm) under aerobic conditions in nutrient broth (NB; see Table S1 at https://doi.org/10.5281/zenodo.18400453 ), adjusted to an optical density of 0.05 at 600 nm (OD 600 ) in pre-warmed NB and further incubated at 37°C and 150 rpm until OD 600 of 0.5–0.6. Then, the bacterial culture was centrifuged (5,000 × g ), and the pellet was re-suspended in NB with 15% (vol/vol) glycerol (Carl Roth, Cat. No. 3783.2). Aliquots were shock-frozen in liquid nitrogen and stored at −80°C. For co-infection experiments, we used the virulent S. suis serotype 2 wild-type strain 10 ( S. suis 10), which was kindly provided by Hilde Smith (formerly Wageningen University and Research, The Netherlands). S. suis was grown on Columbia agar plates supplemented with 7% sheep blood (Oxoid, Thermo Fisher Scientific, Cat. No. PB5008A) overnight at 37°C under aerobic conditions, and cryo-preserved bacterial stocks were prepared at the late exponential growth phase (OD 600 of 1.0) as previously described ( 10 ). Preparation and infection of air-liquid interface (ALI) cultures Primary porcine bronchial (PBEC) and tracheal epithelial cells (PTEC) were isolated from freshly slaughtered swine lungs according to Meng et al. ( 18 ) and as previously described ( 19 ), with some modifications. Lungs were obtained from apparently healthy pigs from a local slaughterhouse (Leine-Fleisch GmbH, Laatzen, Germany). Segments of the main bronchi or trachea were freed from tissue residues and digested in incubation medium (see Table S1 at https://doi.org/10.5281/zenodo.18400453 ) for 48 h at 4°C. PTEC/PBEC were harvested by scraping the cells from the luminal surface using a scalpel blade and cultivated in a T75 collagen I (Merck, Cat. No. C3867)-coated cell culture flask in Airway Epithelial Cell Basal Medium (AEBM; PromoCell, Cat. No. C21260 ) supplemented with several growth factors and antibiotics (Airway Epithelial Cell Growth Medium, AEGM; [see Table S1 at https://doi.org/10.5281/zenodo.18400453 ]) at 37°C and 5% CO 2 in a humidified atmosphere. When cells reached confluence after approximately 5 days, they were detached using 0.05% trypsin-EDTA (TE; stock 0.5%, Thermo Fisher Scientific, Cat. No. 15400054) and were either used for ALI cultures or cryo-preserved in AEGM supplemented with 40% fetal calf serum (FCS; Biochrom, Cat. No. S 0615) and 10% dimethyl sulfoxide (DMSO; Merck, Cat. No. D2650) and stored in liquid nitrogen. For ALI cultures, cells were thawed, cultivated in AEGM until confluence, and then dissociated using 0.05% TE (Thermo Fisher Scientific, Cat. No. 15400054). Subsequently, 2.5 × 10 5 cells were seeded on cell culture inserts with collagen IV (Merck, Cat. No. C7521)-coated polycarbonate membranes (6.5 mm diameter, 0.4 μm pore size; VWR, Cat. No. 734–2742) and incubated under submerged conditions in AEGM at 37°C and 5% CO 2 for 4–5 days. Then, cells were introduced to ALI conditions by adding ALI medium (see Table S1 at https://doi.org/10.5281/zenodo.18400453 ) to the basal compartment only. Under these conditions, cells were maintained for another 3–4 weeks at 37°C and 5% CO 2 to differentiate into a pseudostratified epithelium containing mucus-producing and ciliated cells. Meanwhile, ALI medium in the basal compartment was changed every 2–3 days, and cells were washed once a week with Hank’s Balanced Salt Solution (Thermo Fisher Scientific, Cat. No. 14025100) to remove excessive mucus and dead cells. Epithelial barrier integrity was assessed by measurement of the transepithelial electrical resistance (TEER) using a Millicell ERS-2 voltohmmeter (Merck Millipore). Prior to infection experiments, the differentiated ALI cultures were washed and maintained for at least 1 day without antibiotics. For co-infection experiments, ALI cultures were first infected apically with 10 3 CFU/filter of B. bronchiseptica WT or Δ dnt , respectively, for 4 h. Then, non-adherent bacteria were washed away, and the cells were incubated further for 20 h under ALI conditions at 37°C and 5% CO 2 . Previous experiments have shown that this infection dose is sufficient to colonize the system but does not damage the epithelial cells substantially. Moreover, it has been shown in a porcine precision-cut lung slice (PCLS) model that a period of 4 h is sufficient for bacterial adherence and that B. bronchiseptica can colonize the respiratory epithelium within 24 h, meanwhile reducing the ciliary activity ( 8 ). The next day, ALI cultures were co-infected with 10 7 CFU/filter of S. suis 10 for 4 h, corresponding to previous studies performed with this strain ( 9 , 18 ). Afterward, cells were washed to remove non-adherent Streptococci and incubated further for up to 48 h under ALI conditions at 37°C and 5% CO 2 . After 48 h of co-infection, bacterial growth and colonization reached their maximum, and the epithelial barrier was broken down, which made it necessary to stop the experiment. Samples were taken after 4 h (t 4 ), 24 h (t 24 ), and 48 h (t 48 ) of co-infection ( Fig. 1 ). Fig 1. Open in a new tab Schematic timeline of treatment and sampling during experimental infection of ALI cultures and PCLS with B. bronchiseptica and S. suis . Preparation and infection of PCLS PCLS were prepared from lungs obtained from apparently healthy pigs from a local slaughterhouse (Leine-Fleisch GmbH) as previously described ( 8 ). Ciliary activity was monitored by light microscopy (Leica DMi1; Leica), and slices with at least 80% ciliary activity were chosen for infection experiments. Prior to infection, slices were kept in Roswell Park Memorial Institute (RPMI 1640) medium (Thermo Fisher Scientific, Cat. No. 21,875,034) without antibiotics for at least 1 day. Infection of PCLS was performed in accordance with the infection of ALI cultures described above ( Fig. 1 ). In line with our previous study ( 8 ), the infection dose of B. bronchiseptica WT and Δ dnt , respectively, was 10 4 CFU/slice, and for S. suis 10 7 CFU/slice. Bacterial growth and colonization of S. suis and B. bronchiseptica To distinguish colonies from S. suis and B. bronchiseptica , we used Columbia agar plates supplemented with 7% sheep blood and Oxoid Staph/Strep selective supplement (Thermo Fisher Scientific, Cat. No. SR0070E; referred to as “Strep-agar plate”) for S. suis as previously described ( 8 ). For B. bronchiseptica , we chose Columbia agar plates supplemented with 5% bovine blood, 100 mg/L bacitracin, 1 mg/L lincomycin, and 1 mg/L crystal violet (referred to as “Bordetella-agar plate”). All agar plates were incubated at 37°C under aerobic conditions for 48 h. In order to quantify cell-associated (adherent and/or intracellular) bacteria in ALI cultures, cells were washed, detached by using 0.05% TE (Thermo Fisher Scientific, Cat. No. 15,400,054), and then lysed by adding 1% saponin (Carl Roth, Cat. No. 4185.1) and rigorous pipetting up and down as previously described ( 19 ). Cell lysates were serially diluted and plated on Strep- or Bordetella-agar plates, respectively. To quantify tissue-associated bacteria (surface-adherent and/or intracellular bacteria) in infected PCLS, the slices were washed once and then homogenized in phosphate-buffered saline (PBS; Thermo Fisher Scientific, Cat. No. 14,190,250) using Lysing Matrix D (MP Biomedicals, Cat. No. 1169130-CF) and the FastPrep-24 5 G Instrument (3 × 30 s, 4.5 m/s; MP Biomedicals) as previously described ( 8 ). Afterward, bacterial number in the lysate was determined by serial dilution and replicate plating on Strep- and Bordetella-agar plates, respectively. Similarly, supernatants of infected PCLS or ALI cultures were serially diluted and plated on agar plates to determine the number of CFU in the supernatant. To collect the supernatant from ALI cultures, ALI medium was added to the apical compartment, and ALI cultures were incubated on a horizontal shaker for 5 min at room temperature (RT). Cytotoxicity assay To determine the cytotoxic effects caused by B. bronchiseptica and/ or S. suis , we measured the release of lactate dehydrogenase (LDH) using the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Cat. No. G1780) as previously described ( 8 ). LDH release of infected PCLS/ALI cultures was normalized to uninfected control samples, and the results were expressed as percentage LDH release compared to uninfected samples lysed with 1% (vol/vol, ALI cultures) or 10% (vol/vol, PCLS) Triton X 100 (Carl Roth, Cat. No. 3051.3) in medium, respectively. OD values were measured at 492 nm using SpectraMax i3x (Molecular Devices). Immunofluorescence analysis Prior to whole mount immunofluorescence staining, PTEC/PBEC were washed and fixed with 3.7% (vol/vol) formaldehyde (stock solution 37%; Carl Roth, Cat. No. CP10.1). Then, ALI cultures were incubated for 1–2 h in blocking buffer (see Table S2 at https://doi.org/10.5281/zenodo.18400453 ) at RT. Primary and secondary antibodies (see Table S2 at https://doi.org/10.5281/zenodo.18400453 ) were diluted in antibody dilution buffer (see Table S2 at https://doi.org/10.5281/zenodo.18400453 ), added to the apical compartment, and incubated overnight at 4°C. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, 0.5 μg/mL; Cell Signaling Technology, Cat. No. 4083). Finally, the membrane was cut out and mounted with ProLong Gold Antifade Reagent (Cell Signaling Technology, Cat. No. 9071), as previously described ( 19 ). PCLS were fixed with 4% (vol/vol) phosphate-buffered formalin (Department of Pathology, University of Veterinary Medicine Hannover, Germany), embedded in paraffin (Engelbrecht, Cat. No. 17932a), and sections of 3–4 µm were prepared. Rehydration of paraffin sections, antigen retrieval, and immunofluorescence staining were performed as previously described ( 8 ). Buffers and antibodies used to visualize cilia and bacteria are listed in Table S2 at https://doi.org/10.5281/zenodo.18400453 . Samples were analyzed using the Keyence BZ-X810 fluorescence microscope (Keyence) equipped with the Keyence Plan Apochromat 10×/0.45 or Plan Apochromat 40×/0.95 air objective lens. Image stacks with a z-distance of 0.3–0.4 µm were merged, and brightness, contrast, and colors were adjusted using BZ-X800 Analyzer software (version 1.1.2.4, Keyence). Optical sectioning (confocal-like technology) was applied for structural illumination and high-definition images of ALI cultures. Reverse-transcriptase quantitative real-time PCR (RT-qPCR) The expression of cytokine genes and dnt was analyzed using quantitative real-time PCR following reverse transcription of RNA. For total RNA extraction of ALI cultures, cells were lysed with TRI Reagent (Zymo Research, Cat. No. R2050-1-200) and RNA purification was performed using the Direct-zol RNA MiniPrep Kit (Zymo Research, Cat. No. R2052), including an on-column treatment with DNase I (Qiagen, Cat. No. 79254). RNA concentration and purity (260/280 and 260/230 ratios) were assessed by spectrophotometric analysis using SpectraMax i3x (Molecular Devices). Extracted RNA was stored at -80°C until further analysis. RNA extraction from B. bronchiseptica cultures was carried out in the same way, with an additional lysing step using 0.5 g zirconium beads (Carl Roth, Cat. No. N037.1) and the FastPrep-24 5 G Instrument (2 × 30 s, 6 m/s; MP Biomedicals) prior to RNA purification. Total RNA extraction from PCLS was performed according to the procedures described by Weldearegay et al. ( 20 ). PCLS were stored in RNA later (Merck, Cat. No. R0901) at -80°C until RNA extraction and washed with PBS prior to RNA extraction to remove any residues of RNA later . Slices were first homogenized in RLT buffer (Qiagen; Cat. No. 79,216) using Lysing Matrix D (MP Biomedicals, Cat. No. 1169130-CF) and the FastPrep-24 5 G Instrument (2 × 40 s, 6.5 m/s; MP Biomedicals). One volume of phenol/chloroform/isoamyl alcohol (Carl Roth, Cat. No. A156.1) was added to the lysate, mixed by shaking, and centrifuged at 12,000 × g for 5 min at RT. The aqueous phase was carefully transferred to a new tube, and one volume of chloroform/isoamyl alcohol (49:1; Carl Roth, Cat. No. 6340.1 and Cat. No. T870.1) was added, mixed by shaking, and centrifuged at 12,000 × g for 5 min at RT. The aqueous phase was carefully transferred to a new tube, and purification of RNA was continued using the MagMAX96 for Microarrays Total RNA Isolation Kit (Thermo Fisher Scientific, Cat. No. AM1839) according to the manufacturer’s instructions. RNA concentration and purity (260/280 and 260/230 ratio) were assessed by spectrophotometric analysis using SpectraMax i3x (Molecular Devices), and extracted RNA was stored at −80°C until further analysis. The cDNA was prepared from a total of 500 ng RNA for cytokines and 1 µg for dnt . For transcription of RNA from ALI cultures and bacterial cultures, we used 200 units M-MLV (H-) Point Mutant reverse transcriptase (Promega, Cat. No. M3682), dNTP Mix (10 mM each; Carl Roth, Cat. No. K039.1) and 500 ng Random Primers (Promega, Cat. No. C1181). For no-RT controls, the reverse transcriptase was replaced by RNase-free water. NoRT- and cDNA samples were diluted 1:4 in DNase-free water and stored at −20°C. RNA samples from PCLS were transcribed using the QuantiTect Reverse Transcription Kit (Qiagen, Cat. No. 205311) according to the manufacturer’s instructions, including DNA digestion with the gDNA Wipeout Buffer provided by the kit. For no-RT controls, the Quantiscript reverse transcriptase was replaced by RNase-free water. NoRT- and cDNA samples were diluted 1:10 in DNase-free water and stored at −20°C. Quantitative real-time PCR was performed in 20 µL reaction volumes containing 5 µL of diluted cDNA/noRT-sample, 500 nM gene-specific primers (see Table S3 at https://doi.org/10.5281/zenodo.18400453 ), and 10 µL of QuantiTect SYBR Green PCR Master Mix (Qiagen, Cat. No. 204245) with the Mx3005P Real-Time PCR System (Agilent Technologies). The PCR program consisted of an initial enzyme activation step at 95°C for 20 min, followed by 40 cycles of 30 s denaturation at 95°C, 30 s annealing at 55°C, and 30 s elongation at 72°C, and a melting curve for 1 min at 95°C, 30 s at 55°C, and 30 s at 95°C. All samples were run in duplicates, and non-template as well as noRT-controls were included. Relative gene expression of the cytokines was calculated by normalization to GAPDH transcript levels and to uninfected control samples (ΔΔC q ) ( 21 ), and relative gene expression of dnt was calculated by normalization to the reference gene adk (ΔC q ). Reverse-transcriptase PCR (RT-PCR) Expression of the T-type voltage-gated calcium channels Ca V 3.1 (Gene ID: 100513431, CACNA1G ) and Ca V 3.2 (Gene ID: 110259950, CACNA1H ), which are predicted receptors for the DNT of B. pertussis ( 3 ), was analyzed using PCR following reverse transcription of RNA. RNA isolation and cDNA transcription were performed as described above. Genomic DNA (gDNA) of ALI cultures and PCLS was isolated using the DNeasy Blood and Tissue Kit (Qiagen, Cat. No. 69506) according to the manufacturer’s instructions ( Spin-Column Protocol for Purification of Total DNA from Animal Blood or Cells ). cDNA and gDNA samples from PCLS or ALI cultures infected with B. bronchiseptica WT for 72 h (t 48 ) were then amplified with 0.5 units of HotStarTaq Plus DNA Polymerase (Qiagen, Cat. No. 203605) and 200 nM of each primer (see Table S3 at https://doi.org/10.5281/zenodo.18400453 ) using the Peqlab Advanced Primus 96 cycler (PEQLAB Biotechnologie GmbH, today part of VWR) as follows: initial enzyme activation step at 95°C for 15 min, followed by 45 cycles of 30 s denaturation at 94°C, 30 s annealing at 55°C, and 1 min elongation at 72°C for CACNA1G and annealing at 68°C, and 40 s elongation at 72°C for CACNA1H . Non-template and no-RT controls were included, as well as gDNA from uninfected ALI cultures and PCLS as a positive control. PCR products were loaded on a 2% agarose gel stained with ROTI GelStain (Carl Roth, Cat. No. 3865.2) and visualized on the Intas Gelstick imager (Intas). Enzyme-linked immunosorbent assay (ELISA) The level of the cytokines IL-1α, IL-6, CXCL8, and TNF-α in the supernatant of infected PCLS was measured using porcine DuoSet ELISA kits (R&D Systems, Cat. No. DY680, DY686, DY535, and DY690B) according to the manufacturer’s instructions. Samples were diluted 1:2, 1:10, or 1:20. The Substrate Reagent Pack (R&D Systems, Cat. No. DY999) was used to identify streptavidin-horseradish peroxidase coupled to detection antibodies, and the reaction was stopped after 20 min with 2 N H 2 SO 4 (Carl Roth, Cat. No. X873.1). OD values were measured at 450 nm using SpectraMax i3x (Molecular Devices) and normalized to OD values at 540 nm. The concentration of cytokines (pg/mL) was calculated by interpolation of the normalized OD values to a standard curve using the four-parameter logistic (4-PL) curve-fit model and the software GraphPad Prism version 10.4.1 for Windows (GraphPad Software). The amount of supernatant from infected ALI cultures was not sufficient to perform ELISA. Statistical analysis Data are shown as medians from 3 to 4 independent experiments (in technical duplicates). All statistical analyses were carried out using GraphPad Prism version 10.4.1 for Windows (GraphPad Software). Statistical significance was analyzed using the Mann-Whitney test or Kruskal-Wallis test followed by Dunn’s multiple comparison test with a confidence level of 0.05. Exact P values are listed in Tables S4 and S5 at https://doi.org/10.5281/zenodo.18400453 . RESULTS Expression of dnt in Bordetella bronchiseptica and expression of DNT receptors in the porcine respiratory tract To confirm the absence of dermonecrotic toxin (DNT) production in B. bronchiseptica Δ dnt , we analyzed the expression of dnt in both Bordetella strains grown to the stationary phase in nutrient broth via RT-qPCR, as DNT production has previously been shown in bacterial culture ( 22 ). In comparison to the reference gene adk , dnt was induced 2-fold in the B. bronchiseptica WT, whereas it was reduced 2-fold in the DNT-deficient mutant strain ( Fig. 2A ). Next, we checked whether the calcium channels Ca V 3.1 ( CACNA1G ) and Ca V 3.2 ( CACNA1H ), which are predicted receptors for the DNT of B. pertussis ( 3 ), are expressed in the herein used in vitro models—the porcine respiratory epithelial cells differentiated under air-liquid interface conditions (ALI cultures) and the porcine PCLS. For this, we isolated RNA from ALI cultures and PCLS and performed RT-PCR. The results demonstrated that CACNA1G is expressed in both models ( Fig. 2B ). On the contrary, CACNA1H is indeed present in the genomic DNA of both ALI cultures and PCLS but only expressed in PCLS ( Fig. 2C ). Taken together, in both models, at least one of the receptors needed for DNT binding is expressed, showing that these models are suitable to investigate the role of DNT during infection. Fig 2. Open in a new tab Expression of dnt during bacterial growth and expression of DNT receptors in ALI cultures and PCLS. ( A ) Expression of dnt during growth in nutrient broth was normalized to the expression of adk . The median of three independent experiments is shown. RT-PCR detection of ( B ) CACNA1G and ( C ) CACNA1H expression in ALI cultures and PCLS. M, marker; cDNA, standard RT-PCR reaction using reverse-transcribed RNA; noRT, negative control in which no reverse transcriptase was added to the RT reaction; gDNA, positive control in which genomic DNA was used as a template in the RT-PCR; NTC, non-template control. Colonization of the porcine respiratory epithelium by B. bronchiseptica and S. suis Since we have proven the presence of at least one of the predicted DNT-binding receptors in our in vitro models mimicking the porcine respiratory epithelium, we proceeded to study the interactions between B. bronchiseptica and S. suis as well as the role of DNT in a co-infection scenario. For this, ALI cultures as well as PCLS were initially infected with the virulent B. bronchiseptica wild-type strain (WT), which is positive for DNT, and the isogenic DNT-deficient mutant strain (Δ dnt ), respectively, for 24 h. Subsequently, ALI cultures and PCLS were infected with the virulent S. suis serotype 2 wild-type strain 10 ( S. suis 10). Plating of whole cell/tissue lysates revealed a time-dependent increase of cell- and tissue-associated Bordetellae in ALI cultures as well as in PCLS with a maximum of approximately 4 × 10 8 CFU/mL in ALI cultures and approximately 1 × 10 8 CFU/mL in PCLS after 48 h of infection (t 24 ; Fig. 3A ). The number of cell-/tissue-associated Bordetellae was comparable for the wild-type strain and the mutant ( Fig. 3A ) but was slightly lower when ALI cultures or PCLS were co-infected with S. suis (see Fig. S1 at https://doi.org/10.5281/zenodo.18400453 ). In contrast, the number of Bordetellae in the supernatant of infected ALI cultures or PCLS was slightly increased when they were co-infected with S. suis (see Fig. S2B and C at https://doi.org/10.5281/zenodo.18400453 ), indicating that both bacterial species compete for binding sites and S. suis could displace B. bronchiseptica to some extent. Fig 3. Open in a new tab Association of B. bronchiseptica and S. suis to mono- and co-infected ALI cultures and PCLS. ALI cultures and PCLS were pre-infected with B. bronchiseptica WT and B. bronchiseptica Δ dnt , respectively, for 24 h. Subsequently, ALI cultures and PCLS were infected with S. suis strain 10 for up to 48 h. To calculate the number of cell-/tissue-associated bacteria, cell/tissue lysates were plated on agar plates to determine CFU/mL of ( A ) B. bronchiseptica in mono-infected samples and ( B ) S. suis in mono- and co-infected samples. The median of 3–4 experiments is shown. Significant differences between WT and Δ dnt as well as between S. suis mono-infection and co-infection were analyzed with ( A ) Mann-Whitney test or ( B ) Kruskal-Wallis test followed by Dunn‘s multiple comparisons test (* P < 0.05). The number of cell-associated Streptococci remained almost similar in ALI cultures during the whole infection experiment (approximately 7 × 10 6 CFU/mL) but was significantly increased when cells were pre-infected with B. bronchiseptica WT or Δ dnt , respectively (approximately 2 × 10 7 CFU/mL; Fig. 3B ). Colonization of PCLS by S. suis was time-dependent and enhanced by pre-infection with B. bronchiseptica WT or Δ dnt at t 4 and t 24 ( Fig. 3B ). Interestingly, at t 48 , the number of tissue-associated Streptococci was lower when PCLS were pre-infected with B. bronchiseptica WT or Δ dnt ( Fig. 3B ). In contrast, the number of Streptococci in the supernatant was significantly increased at t 24 and t 48 when PCLS were pre-infected with B. bronchiseptica WT or Δ dnt (see Fig. S2A at https://doi.org/10.5281/zenodo.18400453 ). In ALI cultures, the number of Streptococci in the supernatant was highest at t 4 (approximately 1 × 10 9 CFU/mL) and similar in the supernatant of mono- and co-infected cells. At later time points (t 24 and t 48 ), approximately 4 × 10 8 CFU/mL of S. suis 10 were counted when ALI cultures were pre-infected with B. bronchiseptica WT or Δ dnt , respectively, but only 1 × 10 8 CFU/mL in the supernatant of mono-infected ALI cultures (see Fig. S2A at https://doi.org/10.5281/zenodo.18400453 ). Immunofluorescence analysis confirmed these plating results ( Fig. 4 ). Moreover, visualization of the bacteria revealed that B. bronchiseptica WT and Δ dnt both adhered to the cilia in masses in mono-infected PCLS, whereas when co-infected with S. suis for 24 h, Bordetellae as well as ciliated cells could hardly be detected. On one hand, we assume that cilia are destroyed during co-infection, and as a result, the preferred structures to bind to are missing for B. bronchiseptica . On the other hand, we cannot exclude technical issues during the process of immunofluorescence staining (e.g., loss of bacteria during washing steps). Streptococci were mainly found in the alveolar epithelium of PCLS ( Fig. 4 ). In ALI cultures, B. bronchiseptica preferentially adhered to cilia, as the WT and the mutant strains were almost exclusively found on top of them ( Fig. 4 ). After prolonged infection, almost all cilia were destroyed by B. bronchiseptica , independent of the presence of DNT (see Fig. S3 at https://doi.org/10.5281/zenodo.18400453 ). Notably, immunofluorescence analysis of ALI cultures did not confirm the enhancing effect of B. bronchiseptica pre-infection on the colonization capacity of S. suis that we found by plating of cell lysates. As mentioned above, this could be due to the immunofluorescence staining procedure or due to difficulties in detecting Streptococci via fluorescence microscopy. Thus, the determination of cell-associated bacteria via plating of cell lysates seems to be the more reliable method. Fig 4. Open in a new tab Visualization of bacterial association to mono- and co-infected ALI cultures and PCLS. Immunofluorescence staining of ALI cultures at t 4 (4 h post-co-infection) and PCLS at t 24 . Bordetellae are shown in yellow, Streptococci in purple, cilia (α-tubulin) in red, and nuclei (DAPI) in cyan; bars represent 100 µm. Boxed regions in PCLS images are depicted with a higher magnification in the image to the right. In general, these results show that B. bronchiseptica colonizes the porcine respiratory epithelium in vitro more efficiently compared to S. suis and that DNT has no influence on the colonization capacity of B. bronchiseptica . However, pre-infection with B. bronchiseptica significantly promoted colonization capacity of S. suis , independent of the presence of DNT. Cytotoxic effects of B. bronchiseptica and S. suis on the porcine respiratory epithelium Cytotoxic effects on respiratory epithelial cells upon infection with B. bronchiseptica and/or S. suis were determined by measuring the release of lactate dehydrogenase (LDH) into the supernatant of infected cells. In ALI cultures, the amount of LDH released into the supernatant upon infection with B. bronchiseptica increased over time up to approximately 50% (normalized to cells lysed with 1% Triton X 100) after 72 h of infection (t 48 ; Fig. 5A ) but was similar for the WT and the mutant strain. The highest amount of LDH released upon infection with S. suis was detected after 4 h of infection (approximately 60%, Fig. 5B ), corresponding to the high number of Streptococci in the supernatant at t 4 (see Fig. S2A at https://doi.org/10.5281/zenodo.18400453 ). Cytotoxicity at t 4 was slightly enhanced when cells were pre-infected with B. bronchiseptica WT or Δ dnt , respectively, although the difference was not significant ( Fig. 5B ). At t 24 , mono-infection with S. suis induced only approximately 20% LDH release, whereas co-infection with both pathogens resulted in approximately 75% LDH release ( Fig. 5B ), indicating a synergistic effect of the cytotoxicity induced by both pathogens. At t 48 , the difference between mono- and co-infected ALI cultures was not as obvious (approximately 40% LDH release in mono-infected and 60% in co-infected ALI cultures; Fig. 5B ). Fig 5. Open in a new tab Cytotoxic effects of B. bronchiseptica and/or S. suis and expression of dnt during mono- and co-infection of ALI cultures. ALI cultures were pre-infected with B. bronchiseptica WT and B. bronchiseptica Δ dnt , respectively, for 24 h. Subsequently, ALI cultures were infected with S. suis strain 10 for up to 48 h. ( A and B ) Cytotoxic effects were determined by measuring the release of LDH into the supernatant of infected cells at the indicated times. The results are expressed as percentage LDH release compared to cells lysed with 1% Triton X-100 and normalized to non-infected cells. ( C and D ) Barrier integrity was evaluated by measuring the trans-epithelial electrical resistance (TEER) at the indicated times. The results are expressed as percentage TEER, normalized to non-infected cells. The median of 3–4 experiments is shown. Significant differences between WT and Δ dnt as well as between S. suis mono-infection and co-infection were analyzed with ( A and C ) Mann-Whitney test or ( B and D ) Kruskal-Wallis test followed by Dunn’s multiple comparisons test (* P < 0.05). In addition to the LDH release assay, we defined the detrimental effects of B. bronchiseptica and/or S. suis on the epithelial barrier by measuring the trans-epithelial electrical resistance (TEER), a parameter for the barrier integrity, and compared it to non-infected ALI cultures (set as 100%). At t 4 , infection with B. bronchiseptica WT or Δ dnt alone had no effect on the barrier integrity ( Fig. 5C ), whereas infection with S. suis alone as well as co-infection with both pathogens resulted in a significant drop in the TEER values by approximately 50% ( Fig. 5D ). Interestingly, the barrier integrity recovered 20 h later to almost 100% when infected with S. suis alone. In contrast, infection with B. bronchiseptica WT or Δ dnt caused a time-dependent disturbance of the epithelial barrier ( Fig. 5C ), which was even more pronounced in cells co-infected with S. suis , resulting in TEER values as low as 10% at t 48 ( Fig. 5D ). In accordance with the infection of ALI cultures, we determined the cytotoxic effects of B. bronchiseptica and/or S. suis infection on PCLS by measuring the release of LDH into the supernatant of infected PCLS. Only low amounts of LDH were detected after infection with B. bronchiseptica WT or Δ dnt , approximately 10% after 48 h (t 24 ) and approximately 25% after 72 h of infection (t 48 ; Fig. 6A ). Infection with S. suis alone resulted in similar percentages of LDH release, approximately 5% at t 24 and approximately 35% at t 48 ( Fig. 6B ). At t 4 , almost no LDH was measurable in the supernatants of PCLS infected with B. bronchiseptica or S. suis alone ( Fig. 6B ). However, co-infection with both pathogens led to synergistic cytotoxic effects, indicated by a significant increase of LDH release at t 4 and t 24 ( Fig. 6B ). At t 48 , the amount of LDH was lower in PCLS co-infected with B. bronchiseptica Δ dnt and S. suis compared to PCLS co-infected with B. bronchiseptica WT and S. suis, but in all other samples, no difference between the WT and the mutant strain was detectable ( Fig. 6A and B ). Fig 6. Open in a new tab Cytotoxic effects of B. bronchiseptica and/or S. suis during mono- and co-infection of PCLS. PCLS were pre-infected with B. bronchiseptica WT and B. bronchiseptica Δ dnt , respectively, for 24 h. Subsequently, PCLS were infected with S. suis strain 10 for up to 48 h. ( A and B ) Cytotoxic effects were determined by measuring the release of LDH into the supernatant of infected slices at the indicated times. The results are expressed as percentage LDH release compared to PCLS lysed with 10% Triton X-100 and normalized to uninfected PCLS. ( C and D ) Ciliary activity of uninfected (control) and infected PCLS was monitored at the indicated times by estimating the ciliary beating using light microscopy. The results are expressed as percentage ciliary activity compared to the ciliary activity before infection (set as 100%). The median of 3–4 experiments is shown. Significant differences between WT and Δ dnt as well as between S. suis mono-infection and co-infection were analyzed with ( A and C ) Mann-Whitney test or ( B and D ) Kruskal-Wallis test, followed by Dunn’s multiple comparisons test (* P < 0.05). One special feature of PCLS is the possibility to monitor ciliary beating by light microscopy, which provides information on the fitness and vitality of the tissue. We monitored the ciliary activity during infection and observed that both B. bronchiseptica strains had detrimental effects on the ciliary activity as it was reduced by approximately 60% after 24 h of infection (immediately before co-infection with S. suis (see Fig. S4 at https://doi.org/10.5281/zenodo.18400453 ) and by even 100% after 48 h of infection (t 24 ; Fig. 6C ). In contrast, infection with S. suis alone had almost no effect on the ciliary activity after 4 h and 24 h. Only after prolonged infection with S. suis (48 h), ciliary activity was reduced by approximately 30% ( Fig. 6D ). Additional experiments showed that LDH is only released upon treatment of PCLS with whole bacterial culture of B. bronchiseptica WT but not with cell-free (heat-inactivated) supernatant (see Fig. S5 at https://doi.org/10.5281/zenodo.18400453 ). This indicates that cytotoxic effects of B. bronchiseptica were not induced by a secreted factor but are dependent on the presence of living bacterial cells. Accordingly, ciliary activity was only abolished when PCLS were treated with bacterial culture but not with cell-free (heat-inactivated) supernatant (see Fig. S5 at https://doi.org/10.5281/zenodo.18400453 ). Taken together, B. bronchiseptica infection had harmful effects on the ciliary beating, which were independent of DNT and did not correspond to the cytotoxic effects, which were very low. However, co-infection with both pathogens led to significantly increased damage of the respiratory epithelium. Cytokine response of the porcine respiratory epithelium infected with B. bronchiseptica and/or S. suis During the early stages of bacterial infection, the host usually reacts by expressing pro-inflammatory cytokines, such as IL-1α , IL-6 , CXCL8 , and TNF-α . Thus, we were also interested in studying the pro-inflammatory host cell response during co-infection with B. bronchiseptica and S. suis and whether it is affected by DNT. For this, we studied the induction of respective cytokine genes in ALI cultures as well as PCLS upon infection with B. bronchiseptica and/or S. suis by RT-qPCR. In ALI cultures at t 4 , only minor changes in the expression of IL-1α and TNF-α were observed, but CXCL8 was activated by both pathogens and highly expressed in co-infected epithelial cells (see Fig. S6A at https://doi.org/10.5281/zenodo.18400453 ). At t 24 , expression of IL-1α , CXCL8 , and TNFα was mainly induced by B. bronchiseptica but barely by S. suis ( Fig. 7A ). At t 48 , we detected higher levels of IL-1α , CXCL8 , and TNF-α in co-infected cells compared to cells infected with only one of the pathogens (see Fig. S7A at https://doi.org/10.5281/zenodo.18400453 ). Expression of IL-6 was only induced by infection with S. suis after prolonged infection ( Fig. 7A ; see Fig. S7A at https://doi.org/10.5281/zenodo.18400453 ). Fig 7. Open in a new tab Pro-inflammatory cytokine response toward B. bronchiseptica and S. suis mono- and co-infection of ALI cultures and PCLS. ( A ) ALI cultures and ( B ) PCLS were pre-infected with B. bronchiseptica WT and B. bronchiseptica Δ dnt , respectively, for 24 h. Subsequently, ALI cultures and PCLS were infected with S. suis strain 10 for up to 48 h. Gene expression levels of IL1α , IL-6 , CXCL8 , and TNF-α were analyzed at t 24 (24 h post co-infection) by RT-qPCR and normalized to the expression of GAPDH, and relative fold differences were calculated compared to non-infected cells. ( C ) Cytokine levels in the supernatant of uninfected (control) and infected PCLS were quantified at t 24 using ELISA. The median of 3–4 independent experiments is shown. Significant differences between WT and Δ dnt as well as between S. suis mono-infection and co-infection were analyzed with the Kruskal-Wallis test, followed by Dunn’s multiple comparisons test (* P < 0.05). In contrast to ALI cultures, which consist of epithelial cells only, PCLS provides a composition of different cell types, including epithelial cells, endothelial cells, fibroblasts, as well as some resident immune cells ( 23 , 24 ). Therefore, we assumed that the pro-inflammatory cytokine response upon infection of PCLS differs from the cytokine response in ALI cultures described above. In PCLS at t 24 , the expression of IL-1α , IL-6 , and TNF-α was mainly induced by infection with S. suis and, to a lesser extent, by infection with B. bronchiseptica alone. In contrast, CXCL8 was mainly stimulated by infection with B. bronchiseptica and less after infection with S. suis alone ( Fig. 7B ). Expression of IL-1α , IL-6 , and CXCL8 was induced by both pathogens at t 4 , and all these cytokines were significantly higher expressed when slices were co-infected with B. bronchiseptica and S. suis compared to PCLS infected with S. suis alone. Expression of TNF-α was only slightly induced after mono- and co-infection with S. suis but not by infection with B. bronchiseptica alone (see Fig. S6B at https://doi.org/10.5281/zenodo.18400453 ). At t 48 , expression of all cytokines was only activated in PCLS infected with S. suis alone but not (or only to a lesser extent) in PCLS infected with B. bronchiseptica or slices that were co-infected with both pathogens. Especially, the expression levels of TNF-α were significantly lower in co-infected PCLS compared to S. suis mono-infected PCLS (see Fig. S7B at https://doi.org/10.5281/zenodo.18400453 ). When comparing the expression of cytokines in ALI cultures and PCLS, IL-6 was mainly expressed in the PCLS model, whereas the expression of CXCL8 was primarily activated in ALI cultures. IL-1α was expressed during early infection in PCLS but to a greater extent in ALI cultures after prolonged infection. TNF-α was rarely activated in both models but significantly downregulated after prolonged co-infection of PCLS ( Fig. 7A and B ; see Fig. S7A and B at https://doi.org/10.5281/zenodo.18400453 ). The level of pro-inflammatory cytokines released into the supernatant of infected PCLS was analyzed using enzyme-linked immunosorbent assay (ELISA). At t 24 , we found only low amounts of TNF-α in the supernatant of PCLS and no correlation to the infecting pathogen, which fits the low expression level of TNF-α . In accordance with the high expression of CXCL8 , we found very high levels of CXCL8 in the supernatant of PCLS, especially when infected with B. bronchiseptica . IL-6 was found in high levels in the supernatant, regardless of the infecting pathogen, and IL-1α was mainly released into the supernatant when PCLS were infected with B. bronchiseptica and was higher in co-infected PCLS ( Fig. 7C ). At t 4 , the highest levels of cytokines were found in PCLS that were co-infected with both pathogens (see Fig. S6B at https://doi.org/10.5281/zenodo.18400453 ). At t 48 , the levels of IL-1α, IL-6, and TNF-α were highest in PCLS infected with S. suis alone, which is in accordance with the expression data of these cytokines. Notably, the level of CXCL8 was highest in PCLS infected with B. bronchiseptica alone, whereas it was significantly lower in the supernatant of PCLS infected with S. suis alone or co-infected with both pathogens (see Fig. S7C at https://doi.org/10.5281/zenodo.18400453 ). Taken together, expression and secretion of pro-inflammatory cytokines in our in vitro models during early infection were mainly induced by B. bronchiseptica and during late infection, mainly by S. suis . Co-infection with both pathogens, depending on the time and the analyzed cytokine, had a limited amplifying effect on cytokine expression as well as secretion and, in some cases, even an inhibiting effect. According to our results described above, we did not observe any noteworthy differences between B. bronchiseptica WT and the mutant strain, indicating that DNT is not involved in the activation of the investigated cytokines upon B. bronchiseptica infection. DISCUSSION In a previous study, we clearly demonstrated that B. bronchiseptica can pave the way for S. suis infection ( 8 ). However, we could not yet clarify which virulence factor(s) of B. bronchiseptica is/are involved in this process. Thus, in this study, we set out to investigate the role of the dermonecrotic toxin (DNT) of B. bronchiseptica in mono- and co-infections of the porcine respiratory tract. Furthermore, we included primary porcine respiratory epithelial cells differentiated under air-liquid interface conditions (ALI cultures) in addition to the porcine PCLS. In comparison to PCLS, ALI cultures offer the opportunity to investigate the effects of an infection with respiratory pathogens on the three-dimensional respiratory epithelial barrier and the interactions between the respective pathogen and respiratory epithelial cells in more detail. By using ALI cultures as an additional in vitro model of the porcine respiratory tract, we could show that B. bronchiseptica is able to disrupt the respiratory epithelial barrier, reflected by a decrease of the trans-epithelial electrical resistance (TEER) and the loss of ciliated cells, which might facilitate S. suis colonization and invasion of the respiratory epithelium as it is described for swine influenza virus (SIV) ( 10 ). Our findings are supported by the study of Cao et al., who found that B. bronchiseptica can impair the integrity of the tracheal epithelial cell barrier by cleavage of the E-cadherin adherence junction protein mediated by the bacterial protease HtrA/DegQ ( 25 ). Other research groups identified the adenylate cyclase toxin (ACT) ( 26 ) or the tracheal cytotoxin (TCT) in combination with lipopolysaccharides (LPS) ( 27 ) as the responsible factors for TEER reduction. However, in our study, advanced destruction of the epithelial barrier cannot be correlated with increased S. suis colonization. On the contrary, S. suis adherence was even lower after prolonged infection when TEER measurement indicated a break-down of the epithelial barrier. Interestingly, especially in co-infected ALI cultures, the reduction of the epithelial barrier integrity corresponds to the high amounts of lactate dehydrogenase (LDH) in the supernatant of infected cells, reflecting the cytotoxic activity of both pathogens that results in damage of the epithelial cells. The observed cytotoxic effects are time-dependent but not necessarily linked to increasing bacterial numbers, suggesting that an accumulation of the cytotoxic factors is required to harm the epithelial cells to this extent. In both model systems mimicking the porcine respiratory epithelium, we confirmed the major findings of our previous study: (i) B. bronchiseptica has detrimental effects on ciliated epithelial cells, (ii) B. bronchiseptica can promote adherence and colonization of S. suis , and (iii) B. bronchiseptica can thereby promote cytotoxic effects of S. suis ( 8 ). At this point, it should be noted that we used a different B. bronchiseptica strain ( B. bronchiseptica KM22, isolated from a pig with atrophic rhinitis) compared to our previous study ( B. bronchiseptica 1263/2/18, obtained from a swine herd with unspecific symptoms), indicating that these effects are strain independent. Our findings are in line with a recent publication of Hau et al., who described that pre-inoculation of pigs with B. bronchiseptica KM22 increased nasal colonization with S. suis , although the incidence of S. suis disease was not augmented ( 28 ). B. bronchiseptica KM22 efficiently reduced the ciliary activity in PCLS, as we described it for B. bronchiseptica 1263/2/18 ( 8 ). Notably, additional experiments showed that only bacterial culture, but not cell-free (heat-inactivated) supernatant, is able to reduce ciliary activity. These findings are in contradiction to the long-lasting hypothesis that reduction of the ciliary activity (and destruction of cilia) upon infection with B. bronchiseptica is mediated by TCT, a heat-stable toxin that is released into the supernatant ( 29 ). However, further investigations are needed to clarify these contradictory findings. Moreover, although there seems to be a strong correlation between ciliary reduction by B. bronchiseptica and increased adherence of S. suis , further studies, for example, with a strain unable to impair ciliary activity, are needed to really demonstrate the link between these observations. In the present study, we focused on the role of DNT in mono- and co-infection of the porcine respiratory epithelium. This toxin can be associated with the destruction of nasal conchae observed in atrophic rhinitis of pigs by activation of the small GTP-binding protein Rho, which results in alterations of the cytoskeleton of osteoblasts ( 5 ). Moreover, Brockmeier et al. postulated that DNT might contribute to B. bronchiseptica colonization of the porcine respiratory tract as the mutant strain deficient for DNT (the same strain as used in the herein study) showed a slightly reduced colonization capacity in vivo ( 11 ). Although the effects of DNT on the cytoskeleton of osteoblasts and its involvement in nasal turbinate atrophy are well known, and it is also conceivable that DNT induces alterations in the cytoskeleton of epithelial cells, which might result in an increased bacterial adherence, we could not observe such effects in our in vitro infection models. The mutant strain B. bronchiseptica Δ dnt ( B. bronchiseptica KM24/KB24) showed similar colonization capacities as the wild-type strain, and we did not detect any evidence for cytoskeletal alterations of the epithelial cells. The same was true for co-infection of ALI cultures and PCLS with B. bronchiseptica and S. suis —the wild-type and the mutant strain showed similar colonization capacities, and both strains promoted adherence and colonization of S. suis to the same extent. A comparable outcome was observed in an in vivo co-infection study using a DNT-deficient mutant strain (the same strain as used in the herein study), demonstrating that DNT is not essential for predisposing pigs to infection with toxigenic Pasteurella multocida ( 30 ). In addition, we found that the wild-type strain and the DNT-deficient mutant strain induced similar amounts of LDH release upon infection of ALI cultures and PCLS, respectively, with B. bronchiseptica alone or co-infection with S. suis . This suggests that DNT does not play a role in cytotoxicity of B. bronchiseptica in the herein tested in vitro systems. However, this pathogen possesses several other toxins that were described to contribute to its cytotoxic capacity—TCT, ACT, and LPS ( 26 , 27 , 31 ). Almost 30 years ago, van den Akker described that cytotoxic activity toward epithelial cells appears to be regulated by the two-component signal transduction system BvgAS, and the factor responsible for the cytotoxic effect is not secreted into the culture supernatant, the latter being in accordance with our findings ( 32 ). To confirm the validity of the mutant strain, we performed whole genome sequencing (sequence available in NCBI GenBank under accession number CP18120 ) and analyzed the expression of dnt during growth in bacterial culture medium. Whole genome sequencing confirmed that the dnt gene is interrupted by a gentamycin cassette, and RT-qPCR revealed the expression of dnt in the wild-type strain but not in the mutant strain. A few years ago, Teruya et al. published that the T-type voltage-gated calcium channels Ca v 3.1 and Ca v 3.2 serve as receptors for DNT of Bordetella pertussis ( 3 ), and another group confirmed the presence of these receptors in human lung epithelial cells ( 7 ). Since the DNT of B. bronchiseptica and B. pertussis are nearly identical (99.0% amino acid sequence identity) ( 2 – 4 ), we assumed that the DNT of B. bronchiseptica can bind to these receptors likewise and checked whether they are present in the porcine respiratory tract. Indeed, RT-PCR revealed the expression of CACNA1G (encoding for Ca v 3.1) in both ALI cultures and PCLS, as well as the expression of CACNA1H (encoding for Ca v 3.2) in PCLS. Nevertheless, the direct interaction of B. bronchiseptica DNT with these two receptors remains to be proven in our cell culture systems, and further studies with recombinant DNT are needed to clarify the role of DNT. However, even if a direct interaction of DNT with respiratory epithelial cells could be proven, our results strongly suggest that DNT does not have the same effects on respiratory epithelial cells, as it has been described for osteoblasts. Moreover, it has to be considered that DNT-induced nasal turbinate atrophy seen in vivo is a chronic process that takes several weeks ( 11 ), whereas we analyzed the effects of DNT on respiratory epithelial cells during early infection within a few days only. Finally, we were also interested in investigating the cytokine response during the early stages of co-infection with B. bronchiseptica and S. suis and whether DNT has an effect on this host cell response. We hypothesized that B. bronchiseptica pre-infection might alter the host’s immune response in a way that facilitates S. suis infection. Bordetella has been shown to induce the secretion of pro-inflammatory cytokines (e.g., TNF-α, IL-6, IL-12) as well as the anti-inflammatory cytokine IL-10. In this regard, recognition of LPS by the Toll-like receptor 4 (TLR4) plays an important role ( 33 , 34 ). Other virulence factors that might be involved in the induction/secretion of pro- and anti-inflammatory cytokines are the ACT ( 26 , 35 ) and the TCT ( 27 ). Notably, to our knowledge, no studies on the role of DNT in modulation of the host’s immune response, and no investigations on the immune response in the porcine respiratory tract upon infection with B. bronchiseptica have been reported so far. In accordance with the literature, we found that B. bronchiseptica infection led to a relatively fast pro-inflammatory cytokine response. In contrast, S. suis infection barely induced the expression and secretion of pro-inflammatory cytokines in ALI cultures or PCLS. Similar results were described for S. suis infection of newborn pig tracheal epithelial (NPTr) cells, as IL-1α, IL-6, IL-8 ( CXCL8 ), and VCAM1 were not expressed ( 14 , 36 ). Interestingly, another study reported no induction of TNFα but intermediate expression of IL-6 and high expression of IL-8 ( CXCL8 ) upon infection of the same cells ( 37 ). All studies used highly virulent serotype 2 strains ( S. suis 31533 or S. suis P1/7) but different bacterial incubation times. Thus, induction of IL-6 and IL-8 ( CXCL8 ) expression by S. suis might be time-dependent and only activated after prolonged infection, which we also observed in our PCLS infection model. A difference in the temporal dynamics of the host’s immune response was also reported in our previous study, in which we showed that expression of pro- and anti-inflammatory cytokines peaked early during infection of PCLS with a non-virulent and a moderately virulent S. suis strain, whereas gene expression peaked much later (24 h post-infection) when infected with the highly virulent strain 10 (the same strain as used in the herein study) ( 20 ). Notably, although expression of CXCL8 increased after prolonged infection, CXCL8 levels in the supernatant of PCLS mono- and co-infected with S. suis were significantly reduced. This might be explained by cleavage of CXCL8 by a serine protease of S. suis as described by Vanier et al. ( 38 ). Co-infection of PCLS with B. bronchiseptica and S. suis resulted in a higher expression of IL-1α , IL-6 , and CXCL8 during early infection compared to the levels after mono-infection with either of the pathogens. Similar results were described for co-infection of NPTr cells with SIV (H1N1) and S. suis ( 36 , 37 ), Glasserella parasuis and S. suis ( 14 ), or Mycoplasma spp . and S. suis ( 13 ), whereby the authors called it an additive rather than a synergistic effect. Although these and our results were obtained from in vitro infection experiments, one can hypothesize that the enhanced inflammatory immune response might contribute to aggravated streptococcal disease, as an immune system that is already dealing with one infection may not be as resilient to a secondary infection. In in vivo infection experiments, Hau et al. did not observe a higher incidence of S. suis disease in pigs pre-infected with B. bronchiseptica . Nevertheless, the authors stated that B. bronchiseptica pre-infection might enhance streptococcal disease when pigs are additionally immunocompromised or stressed ( 28 ). It has to be noted that, with regard to the host’s immune response, PCLS mimic the in vivo situation more closely than ALI cultures, as PCLS contain several resident immune cells, such as antigen-presenting cells, macrophages, and T cells ( 23 , 24 ), and show characteristic responses to pro-inflammatory stimuli ( 39 ), whereas ALI cultures consist of epithelial cells only. A recent study on the anti-pertussis response of human airway epithelium revealed a dose-dependent secretion of IL-6 and CXCL8 after infection with B. pertussis only, while an additional treatment with IFN-γ, IL-1β, and TNF (cytokines that are secreted by immune cells in response to B. pertussis ) resulted in secretion of several chemokines, indicating that immune cells are required for an effective immune response ( 40 ). Nevertheless, epithelial cells play a critical role in the innate immune response through the secretion of mucin, antimicrobial peptides, and reactive oxygen species, as well as cytokines and chemokines, which in vivo recruit and activate other immune cells ( 41 , 42 ). Thus, both models complement each other perfectly and are most suitable to investigate host-pathogen as well as pathogen-pathogen interactions. Another point that should be considered when working with primary cell cultures is that both immune cells and epithelial cells may already have been activated by previous infections or other harmful substances during the pig’s lifetime, even if the pigs were apparently healthy at the time of slaughter. Although this more closely reflects the in vivo situation where several pathogens might colonize the pig’s respiratory tract sequentially or even simultaneously, or airways might be already impaired by bad air conditions, it must be taken into account when interpreting in vitro data from primary cell cultures. Conclusion In this study, we analyzed the role of DNT during mono- and co-infections of the porcine respiratory tract in vitro and found no evidence that DNT contributes to colonization capacity or cytotoxic activity of B. bronchiseptica, nor does it facilitate co-infection with S. suis in any way. Furthermore, we are the first to describe the pro-inflammatory cytokine response in the porcine respiratory epithelial cells in vitro upon mono-infection with B. bronchiseptica and co-infection with S. suis. B. bronchiseptica activated an early pro-inflammatory immune response, independent of DNT, whereas expression and secretion of cytokines peaked late upon infection with S. suis . Co-infection with both pathogens resulted in increased levels of the herein analyzed cytokines, suggesting that an enhanced inflammatory response might aggravate streptococcal disease. Nevertheless, further investigations are necessary to analyze the complex interactions between the two pathogens as well as their interactions with host cells and the host’s immune system in more detail. The porcine PCLS model, as well as porcine respiratory epithelial cells differentiated under air-liquid interface conditions (ALI cultures), represents excellent in vitro systems to study these interactions. ACKNOWLEDGMENTS This work was financially supported by the Deutsche Forschungsgemeinschaft (DFG) under grant VA239/7-2. The funder had no role in the design of the study, in the collection, analyses, or interpretation of the data, in the writing of the manuscript, or in the decision to publish the results. The authors thank Leine-Fleisch GmbH (Laatzen, Germany) for providing swine lungs. 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All supplemental figures and tables are also available in the Zenodo data repository ( https://doi.org/10.5281/zenodo.18400453 ). Raw sequence data of Bordetella bronchiseptica KM24 whole genome sequencing have been deposited in NCBI Sequence Read Archive under accession number PRJNA1214268 and the genome of KM24 is available in NCBI GenBank under accession number CP181209 . 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