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Characterization of biofilm formation and induction of apoptotic DNA fragmentation by nontypeable Haemophilus influenzae on polarized human airway epithelial cells
Microbial Pathogenesis 141 (2020) 103985
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Characterization of biofilm formation and induction of apoptotic DNA fragmentation by nontypeable Haemophilus influenzae on polarized human airway epithelial cells
T
Buket Baddala,b,∗ a b
Department of Medical Microbiology and Clinical Microbiology, Faculty of Medicine, Near East University, 99138, Nicosia, Cyprus Microbial Pathogenesis Research Group, DESAM Institute, Near East University, Nicosia, Cyprus
A R T I C LE I N FO
A B S T R A C T
Keywords: NTHi Biofilm formation Calu-3 cells Primary bronchial epithelium Apoptosis
Nontypeable Haemophilus influenzae (NTHi) is a common airway commensal and opportunistic pathogen that persists within biofilm communities in vivo. Biofilm studies so far are mainly based on assays on plastic surfaces. The aim of this work was to investigate the capacity of clinical NTHi strains to form biofilm structures on polarized Calu-3 human airway epithelial cells and primary normal human bronchial epithelial cells and to characterize the biofilm architecture. Formation of adherent NTHi biofilms post colonization of host cells at multiple time-points was evaluated using confocal laser scanning microscopy and electron microscopy. NTHi biofilms were analyzed in terms of biofilm height and presence of extracellular matrix components, and their apoptotic effects on epithelial cells were measured by TUNEL assay. Strain Fi176 was observed to form robust biofilms on airway epithelia over time, while disrupting the integrity of Calu-3 monolayer by 72 h of co-culture. NTHi biofilms were observed to induce apoptotic DNA fragmentation in host cells at 24 h post infection. Biofilm formation on cell monolayers by Fi176ΔpilA strain was markedly reduced compared to WT strain. Biofilm inhibition and disruption assays by crystal violet staining indicated that DNA and proteins are part of NTHi biofilms in vitro. Our findings highlight critical stages of NTHi pathogenesis following host colonization and provide useful biofilm models for future antimicrobial drug discovery investigations.
1. Introduction Nontypeable Haemophilus influenzae (NTHi) is a Gram-negative, unencapsulated opportunistic pathogen that commonly colonizes the human nasopharynx. While colonization can be asymptomatic, NTHi also establishes a pathogenic lifestyle within the host respiratory mucosa when host immune defenses are compromised or when bacterial virulence is enhanced [1]. NTHi is responsible for a wide range of respiratory tract infections including sinusitis, acute/chronic/recurrent otitis media (OM) and community-acquired pneumonia, and is implicated in majority of chronic infections of the lower respiratory tract such as cystic fibrosis (CF) and exacerbations of chronic obstructive pulmonary disease (COPD) [2]. The emergence of NTHi as a potential pathogen has been primarily observed after the introduction of H. influenzae serotype b (Hib) conjugate vaccine [3]. The high genetic diversity among strains is one of the main hurdles for the development of effective vaccines against NTHi, while the need for further efforts to understand the pathogenicity mechanisms, in order to identify novel
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antigens with cross-protective capacity, remains a high priority [4]. A critical first step in NTHi infections is adherence to host airway epithelial cells, which has been characterized in a variety of in vitro and in vivo experimental models [5–9]. Interactions of bacteria with host cells including adhesion, invasion, intracellular replication, intracellular trafficking, biofilm formation, bacterial metabolic adaptation, changes in stress response and evasion of host immunity have been the focus of NTHi research [10]. Similar to multiple other pathogenic bacteria affecting the respiratory tract such as Streptococcus pneumoniae [11], Pseudomonas aeruginosa [12] and Moraxella catarrhalis [13], NTHi infections are also complicated by the ability of the pathogen to rapidly establish biofilms, particularly in the middle ear [14–16]. Formation of biofilm communities is considered a virulence strategy due to their inherent antibiotic-resistant nature as well as their capacity to evade host defense factors [17]. Indeed, NTHi biofilms have recently been shown to be central to the pathogenesis of pulmonary infections such as bronchitis, CF and COPD [18]. Recent in vitro studies have confirmed that NTHi biofilms exhibit high antibiotic resistance [19].
Department of Medical Microbiology and Clinical Microbiology, Faculty of Medicine, Near East University, Nicosia, 99138, Cyprus E-mail address: [email protected].
https://doi.org/10.1016/j.micpath.2020.103985 Received 23 September 2019; Received in revised form 14 January 2020; Accepted 15 January 2020 Available online 20 January 2020 0882-4010/ © 2020 Elsevier Ltd. All rights reserved.
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scanning electron microscopy (JEOL JSM 6010LA InTouchScope).
Biofilm formation by the bacterial pathogen have been investigated in diverse in vitro static [20] as well as continuous-flow biofilm systems [21] and animal models of OM [22]. Using these systems, roles for lipooligosaccharides (LOS) sialylation, presence of phosphorylcholine, bacterial and eukaryotic DNA have been proposed to play a role in the development of biofilms [23]. More recently, transformed cell lines have been utilized to provide a more close-to-in vivo models of human airways, where bacterial biofilms have been grown on immortalized epithelial cell monolayers [24,25]. The use of physiologically relevant models of infection is vital to our understanding of infectious processes by pathogens. The present study assessed the capacity of clinical NTHi strains to form biofilms in vitro during prolonged infections on Calu-3 airway epithelial cells as well as primary bronchial epithelial cells with a pseudostratified epithelium phenotype, and investigated the characteristics of NTHi biofilms in terms of matrix components and changes in host cell physiology over time.
2.2. Bacterial culture and infection conditions NTHi strains Fi176 and Fi162 are clinical isolates obtained from patients with otitis media [27]. Strain R2846 was also isolated from the middle ear of a child with otitis media [28]. Deletion of pilA was performed to generate Fi176ΔpilA strain by allelic replacement of the entire coding sequence with an erythromycin resistance as described previously [29]. The genomic sequence of NTHi wild-type strain 176 was obtained using whole-genome sequencing [30]. All NTHi strains were reconstituted from frozen glycerol stock cultures, propagated on PoliVitex chocolate agar (bioMérieux) and incubated overnight at 37 °C with 5% CO2. For infection studies, NTHi was grown in brain heart infusion (BHI) broth (Difco Laboratories) supplemented with 10 μg/ml each of hemin (Fluka Biochemika) and nicotinamide-adenine nucleotide (NAD) (Sigma-Aldrich) until the optical density at 600 nm (OD600) reached 0.5. Bacteria were pelleted and resuspended in infection medium comprised of unsupplemented BEBM (for NHBE cells) and DMEM/F12 without penicillin/streptomycin, supplemented with %2 FBS (for Calu-3 cells) to prepare the inoculum. For infections, the inoculum was added to the apical surface of the cultures at multiplicity of infection (MOI) of 100:1, and incubated for 1 h at 37 °C and 5% CO2. Medium-only controls were used. After 1 h incubation, the inoculum was removed, and the apical surface was gently rinsed three times with 500 μl PBS in order remove any nonadherent bacteria. All infections were undertaken in triplicates. For biofilm formation studies, infected cultures were incubated for indicated periods of time and antibiotic-free basolateral medium was replaced every two days.
2. Materials and methods 2.1. Cell culture Calu-3 cells (ATCC No. HTB-55) were expanded in 75-cm2 flasks using DMEM/F12 supplemented with 1% MEM non-essential amino acid solution (Gibco), %10 Fetal Bovine Serum (FBS, Gibco) and 1% penicillin/streptomycin (Gibco). Cells were dissociated with %0.25 Trypsin-EDTA (Gibco) and seeded on semipermeable polyester membrane transwell supports (12-mm diameter, 0.4-μm pore size; Costar) at a cell density of 5 × 105 cells/ml. Medium (0.5 ml) was added to the basolateral chamber. When confluence was reached (day 4), medium on the apical surface was removed to produce air-liquid interface (ALI). Medium in the basolateral chamber was replaced every other day. Cells were cultured for an additional 10 days and transepithelial resistance (TEER) measurements were taken using an epithelial volt ohmmeter (EVOM2; World Precision Instruments) to ensure cell polarization. Transwell inserts with TEER values > 1000 Ω/cm2 were used for experimentation. To prepare cultures for infection, media with antibiotics were removed and cells were apically and basolaterally washed with phosphate-buffered saline (PBS, Sigma-Aldrich). After three washes with PBS, 0.5 ml antibiotic-free culture medium was added to basolateral chamber and cells were incubated with antibiotic free media for 24 h. For primary cell cultures, primary normal human bronchial epithelial (NHBE) cells (Clonetics-BioWhittaker, San Diego, CA) were used as previously described [26]. Briefly, cells were expanded in 75-cm2 flasks, using bronchial epithelial basal growth medium (BEBM; Lonza) supplemented with the BEGM (bronchial epithelial cell growth medium) BulletKit (Lonza) as recommended by the supplier at 37 °C in 5% CO2 until ~80% confluence, and used between passages 1 and 3. Cells were dissociated using StemPro Accutase cell dissociation reagent (Gibco) and were seeded onto semipermeable membrane supports (12mm diameter, 0.4-μm pore size; Costar) that were previously coated with a solution of collagen type I from rat tail (Gibco) at a concentration of 0.03 mg/ml. The cells were seeded at a density of 105 cells per well using bronchial air-liquid interface (B-ALI) medium (Lonza) supplemented with the B-ALI BulletKit (Lonza). When confluence was reached, the apical medium was removed and ALI was established to trigger differentiation. Cells were maintained at ALI for at least 28 days prior to use in biofilm assays, with the basolateral medium being changed every second day, to ensure a differentiated cell population. The apical side was rinsed with PBS every week to remove excess mucus production. Cell polarity and tight junction (TJ) barrier function were verified by TEER measurements using an epithelial volt ohmmeter. Cultures with TEER values of 800 Ω/cm2 were used for experimentation. Mucus secretion and cilia development were monitored by immunofluorescence microscopy (Zeiss LSM 710) and confirmed by
2.3. Immunofluorescence staining and confocal microscopy For each infection time-point, biofilm-containing transwell inserts were fixed with 4% paraformaldehyde. Membranes were permeabilized and blocked with 3% bovine serum albumin and 0.1% Triton X-100 in PBS. Incubation with rabbit anti-total NTHi serum (in-house), 1:5000, biotinylated soybean agglutinin (SBA, Vector Laboratories), 1:500, Streptavidin Alexa Fluor 594 Conjugate (Invitrogen), 1:1000, Alexa Fluor 647 phalloidin (Invitrogen), 1:1000 and Hoechst 33342 (Invitrogen), 1:10000 were performed either overnight at 4 °C or for 2 h at room temperature (RT). Samples were mounted using ProLong Gold Antifade Mountant (Invitrogen) and analyzed by confocal microscopy using a Zeiss LSM 710 confocal microscope. For z-stack imaging, at each time point, three stacked images at x100 magnification were obtained. Each of the stacked images were selected from an apical surface area at random. Three-dimensional rendering was performed using Imaris software (Bit-Plane, Inc.). Biofilm height on airway epithelia was measured using Zen 2.3 Image Analysis Software 3D Rendering Module. For apoptosis assays, cells were stained using ClickiT™ TUNEL Alexa Fluor™ 488 Imaging Assay (Invitrogen) according to manufacturer's instructions. Uninfected cells were used as negative controls and DNase I treated cells were employed as positive controls.
2.4. Electron microscopy All samples were fixed in 2.5% glutaraldehyde and 2.5% paraformaldehyde in 0.1 M sodium cacodylate buffer overnight, washed in buffer, and secondarily fixed in 1% osmium tetroxide in cacodylate buffer for 1 h. Samples were washed in water and block stained with 1% uranyl acetate for 1 h. After an hour, samples were dehydrated with ethanol using progressively increasing concentrations and dried by the critical point method using CO2 in a Balzers Union CPD 020 (BAL-TEC AG), sputter coated with gold in a Balzers MED 010 unit, and observed with a JEOL JSM 6010LA InTouchScope scanning electron microscope. 2
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orthogonal image sections in Fig. 2C. As morphological evidence by confocal microscopy suggested biofilm structures forming on cell monolayers, scanning electron microscopy (SEM) was employed to provide a more direct visualization of the biofilm matrix. Uninfected cells are shown in Fig. 3A. SEM imaging at 1 hpi revealed attachment of bacteria to apical surface of cells (Fig. 3B), while bacteria were observed to be actively dividing and forming biofilm structures by 24 h (Fig. 3C). At 72 hpi, NTHi biofilms were observed over airway microvilli, in which bacterial cells were enmeshed with exopolysaccharide matrix (white arrow) of the biofilm and host cell mucus as evident in Fig. 3D.
2.5. Biofilm formation assay on plastic For biofilm formation assays, all bacterial strains from overnight cultures were diluted 1:100 and incubated statically in BHI on plastic 24-well plates at 37 °C with 5% CO2. Uninoculated BHI only controls were used. After 24 h, the wells were gently washed once with 1 ml sterile PBS and allowed to dry for 10 min. Bacteria were stained with 1 ml of filter-sterilized 0.2% crystal violet (Sigma) and incubated for 30 min at RT. Crystal violet was removed from the wells, followed by two washes with 1 ml PBS. The dye was extracted by adding 1 ml of 96% ethanol to each well and incubating for 30 min at RT. The absorbance was measured at 540 nm using a Tecan Infinite 200 PRO plate reader. Inhibition of biofilm formation was assessed by adding 0.1 mg/ ml proteinase K or 2 U/mL DNase to bacteria in wells at the beginning of the incubation in the plates and quantified as above. For biofilm disruption assays, biofilms were grown on 24-well plates for 24 h, nonattached cells were removed, and then biofilms were subjected to treatment with 0.1 mg/ml proteinase K or 2 U/mL DNase. Cultures were further incubated for 24 h with the enzymes and were subjected to staining with crystal violet. Absorbance measurements were taken as described above.
3.2. Biofilm formation on primary normal human bronchial epithelial (NHBE) cells In order to confirm observations obtained with Calu-3 cell model, a fully differentiated airway epithelial model comprising of primary NHBE cells was used. When grown at ALI and provided necessary growth supplements, NHBE cells form a pseudostratified epithelium with TJs and adherence junctions (AJs), and contain a high population of ciliated cells (Fig. S1). The airway epithelium also exhibits TEER, mucus secretion and mucociliary activity, and therefore represents an environment similar to in vivo airway lumen. Differentiated NHBE cultures were infected apically with strain Fi176 and biofilm formation capacity at ALI was monitored in a time-course of infection by confocal microscopy. NTHi (red) were labelled with anti-total NTHi serum, bronchial epithelium was stained with phalloidin (green) and nuclei were counterstained with Hoechst 33342 (blue). As biofilm structures started being apparent at 24 hpi on Calu-3 cells, NHBE cells were fixed initially at this time-point and analyzed. At 24 hpi, bacterial aggregates (red) were observed mostly in between NHBE cell boundaries (green) on the apical side with a measured thickness of 4 μm (Fig. 4A). A progressive increase in biofilm mass forming on airway epithelia was observed at 48 and 72 hpi, in which biofilm thickness was 8 μm and 14 μm, respectively (Fig. 4B and C).
2.6. Statistical analysis All data are expressed as mean standard deviation (SD) of the triplicate experimental data. A two-tailed Student's t-test was used to determine the differences in biofilm formation between the control and each group. P value of < 0.05 was taken as significant. 3. Results 3.1. Biofilm formation on Calu-3 airway cells To investigate the capacity of NTHi to form biofilm on airway cells, Calu-3 lung adenocarcinoma cell line in a transwell system was used. Calu-3 cells were cultured on porous membrane at ALI to trigger cell polarization and formation of TJs (Fig. 1). Polarized monolayers were synchronically infected with NTHi strain Fi176 and monolayers were analyzed by immunofluorescent staining and confocal microscopy at 1 h, 24 h and 72 h post infection (hpi). Bacteria (red) were labelled with anti-total NTHi serum and epithelium was stained with phalloidin (gray) to visualize F-actin filaments. At 1 h post inoculation, adherent NTHi were found in low numbers scattered over the cell surface on the apical side of the monolayer (Fig. 2A). After 24 h of incubation, bacteria were observed to adapt to the host epithelium and multiply in number, forming microcolonies on airway epithelia (Fig. 2B). Importantly, bacteria were not exposed to any media apically in this model, mimicking the in vivo lung environment. No intracellular bacteria were detectable by microscopy. A drastic increase in the propensity of NTHi to form biofilms was evident at prolonged infection time-points, with aberrant rearrangements of the host cytoskeleton at 72 hpi as shown in
3.3. Induction of apoptosis in airway cells by NTHi biofilms The role of biofilm phenotype in apoptosis of infected Calu-3 cells was determined by terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) staining. TUNEL assay detects apoptotic cells that have undergone extensive DNA degradation during the late stages of apoptosis and is based on the ability of TdT to label blunt ends of double-stranded DNA breaks. Calu-3 cells infected with NTHi strain Fi176 at MOI 100:1 were analyzed at 1, 2, 4, 6, 24, 48 and 72 hpi by confocal laser scanning microscopy (CLSM) for the appearance of apoptotic cells. While apoptotic cells were not observed until 6 hpi (Fig. 5A–D), CLSM imaging showed that biofilms formed by strain Fi176 induced apoptosis in host cells at 24 h of infection, after which the number of TUNEL-positive cells increased exponentially at 48 and 72 hpi (Fig. 5E–G). Mock infected cells were used as negative controls
Fig. 1. Transwell model of Calu-3 airway epithelium and NTHi time-course infection. Calu-3 cells were cultured on 12 mm transwell permeable inserts at air-liquid interface to establish polarized monolayers. Polarized cell layers were infected with NTHi at a multiplicity of infection 100:1 and biofilm formation was monitored at 1, 24 and 72 hpi. 3
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Fig. 2. NTHi progressively forms biofilms on Calu-3 airway epithelial cell monolayers. Representative en face and orthogonal sections imaged by confocal laser scanning microscopy are shown (magnification, x100). (A) Few bacterial cells (red) attached on the apical surface of cells (gray) at 1 hpi. (B) At 24 hpi, NTHi forms microcolonies on host cell surface. (C) At 72 hpi, NTHi forms biofilms on airway epithelia and induces aberrant changes in host cell architecture. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) Fig. 3. Scanning electron microscopy of NTHi biofilms grown on Calu-3 monolayers. (A) Uninfected Calu-3 monolayers with microvilli 14 days post culture at air-liquid interface. (B) Bacterial cells attaching to host cell surface clearly visible at 1 hpi. (C) NTHi microcolonies and biofilm structures forming on cells at 24 hpi. (D) Thick biofilms observed over host cell microvilli with apparent biofilm exopolysaccharide (white arrows). Magnification shown for each section separately.
plates were employed. Treatment of preformed bacterial biofilms with 0.1 mg/ml proteinase K and 2 U/mL DNase separately caused significant dispersal of NTHi biofilms when compared to untreated wells (Fig. 7A), indicating that their matrix contains proteins and DNA important to their maintenance. Similarly, in biofilm inhibition assays, where bacteria were inoculated and incubated with the enzymes for 24 h, a significant reduction in biofilm-forming capacity of bacteria was observed (Fig. 7B). These data suggest that extracellular DNA and proteins are part of the NTHi biofilm matrix.
where no apoptosis was observed, whereas DNase I treated cells served as positive control for TUNEL assay in which enzyme treated host cells exhibited an apoptotic population. 3.4. Composition of NTHi biofilm matrix To be able to gain insights into the composition of NTHi biofilm matrix forming on Calu-3 airway epithelia, SBA was used to detect the presence of matrix exopolysaccharides in a more physiological model compared to microtiter assays, which are commonly performed on plastic surfaces. SBA is a lectin known to preferentially bind to oligosaccharide structures with terminal α- or β-linked N-acetylgalactosamine. In this assay, epithelial cell nuclei were labelled with Hoechst 33342 (Fig. 6A) and bacteria were labelled with anti-total NTHi serum (Fig. 6B). Immunofluorescence imaging of NTHi biofilms at 72 hpi revealed positive labeling by SBA (Fig. 6D), which indicates that NTHi biofilm matrix consists of sugar moieties such as oligosaccharide structures with terminal N-acetylgalactosamine as shown by the co-localization (yellow) of SBA staining (green) and bacterial biofilm (red) (Fig. 6E). For further analysis of the biofilm matrix, biofilm inhibition and disruption assays were set up for strain Fi176 using DNase I and proteinase K. As the treatment of host cells with these proteolytic and DNAdegrading enzymes would compromise the viability of eukaryotic cells, standard biofilm assays with crystal violet staining on plastic 12-well
3.5. Biofilm formation capacity of clinical NTHi strains A number of factors have been described to contribute to biofilm formation by bacterial pathogens. To be able to confirm microscopy results with a quantitative in vitro system, crystal violet assays have been performed. All three clinical isolates were compared for their biofilm forming capacity on plastic plates for 24 h. Crystal violet staining, a well-accepted bioassay, confirmed that strain Fi176 forms biofilms on plastic as it does on epithelial monolayers. Importantly, strain Fi176 formed significantly more biofilm biomass than strains Fi162 and R2846 (Fig. 7C). To be able to test the contribution of NTHi type IV pilus, in particular the major protein subunit PilA, to biofilm formation, strain Fi176 and its isogenic mutant strain Fi176ΔpilA were compared by crystal violet assays. A statistically significant difference between the biomass formed by strain Fi176 WT and Fi176ΔpilA was 4
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Fig. 4. NTHi forms thick biofilms on differentiated primary normal human bronchial epithelium. Confocal microscopy images of en face and orthogonal sections are shown (magnification, x40). (A) At 24 hpi, NTHi (red) forms microcolonies on NHBE cells (F-actin: green, nuclei: blue) with a height of 4 μm. (B) NTHi rapidly grows on airway epithelia and forms biofilm structures up to a height of 8 μm by 48 hpi. (C) Progressive increase in biofilm mass with a height of 14 μm is observed by 72 hpi. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
mainly adhesion, invasion and host evasion have been thoroughly studied using primary cell models of infection, limited data exists for biofilm formation on airway epithelia. In one of the first reports on NTHi biofilm formation on epithelial cells, NTHi strain 2019, a clinical strain from a COPD patient, was used and biofilm structures were observed on Calu-3 airway cells cultured on transwell inserts up to 5 days [24]. In a study by West-Barnette et al., immortalized human HMEEC-1 middle ear epithelial cells and 16HBE14o− airway epithelial cells were cultured at ALI on permeable membrane inserts, and electron microscopy revealed biofilm formation on apical surface by day 2 by NTHi strain 2019 [25]. In our study, clinical OM strain Fi176 formed significant biofilms on Calu-3 airway cells up to 3 days in co-culture. In accordance with a previous study which demonstrated that NTHI strain 86-028NP, a pediatric OM isolate, established biofilms on confluent monolayers of chinchilla middle ear epithelial cells and induced epithelial cell membrane ruffling and compromise of the epithelial cell membrane integrity [32], our results indicated that when NTHi biofilms form on airway cells at 24 h, the epithelial architecture loses integrity and cells undergo apoptosis as apparent by DNA fragmentation. The number of apoptotic host cells increases consistently up to 72 h of infection. Primary ciliated epithelial cells infected with NTHi have been previously characterized in terms of cell invasion and intracellular survival [33,34], however to our knowledge this is the first study to demonstrate NTHi biofilm forming on differentiated primary NHBE cells up to 72 h and with a biofilm height of 14 μm. The literature on NTHi biofilms have so far described some of the important factors which play a role in biofilm formation as well as the constituents of the biofilm matrix using in vitro microtiter or chamber slide, and in vivo chinchilla model of infection. Work from several
observed, confirming the role of PilA in NTHi biofilms (Fig. 7C). Importantly, these observations were confirmed by CLSM imaging of biofilms formed on Fi176ΔpilA infected Calu-3 monolayers, which in comparison to biofilms formed by WT strain were markedly reduced (Fig. 6C).
4. Discussion NTHi is a commensal bacterial pathogen most commonly associated with asymptomatic carriage, but under predisposing conditions develops strategies to cause persistent infections of the respiratory tract. There is a well-documented contribution of biofilm formation by NTHi to the chronic nature of OM, CF and COPD in the bronchial airway or middle ear [18,31]. Although a plethora of in vitro studies have been performed for elucidation of the role of biofilms in disease and their functional characteristics, majority of the work has been based on measurements of biofilm mass on plastic microtiter plates coupled with crystal violet staining. While these assays are highly informative when comparing a large number of clinical isolates for their biofilm forming capacity and are relatively low cost, they fail to provide physiologicallyrelevant conditions such as those found in the microenvironment of human airways. In this study, we have developed biofilm models for clinical NTHi strains grown on Calu-3 airway epithelial cells and on more physiologically-relevant in vitro differentiated primary human bronchial epithelium, and show that NTHi forms biofilms on airway epithelia over time with oligosaccharide structures with terminal α- or β-linked N-acetylgalactosamine (GalNAc), extracellular DNA and proteins as part of biofilm matrix. Although the interactions of NTHi with human epithelial cells, 5
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Fig. 5. NTHi biofilms induce apoptosis in host cells. Calu-3 cells were infected with NTHi strain Fi176 for 72 h and cells were analyzed by TUNEL assay. Bacteria are labelled in red, host cell nuclei in blue and TUNEL-positive cells in green. (A–D) No apoptotic cells were observed at 1–6 hpi. (E–F) NTHi induced apoptotic DNA fragmentation in host cells at 24 hpi. Apoptotic cells increased in number by 72 hpi (magnification, x10). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
a strain-specific manner [38]. In our study, biofilm inhibition and dispersal assays using standard crystal violet staining have shown that NTHi biofilms contain DNA and proteins, which is consistent with previous findings. In their study, Domenech et al. reported positive labeling for NTHi biofilm exopolysaccharides with only concanavalin A lectin, with α-Man and α/β-Glc specificity, in NTHi biofilms grown in glass-bottomed dishes [20]. In contrast to these findings, our results show positive staining of biofilms grown on Calu-3 monolayers with
groups have characterized the proteome of NTHi in biofilms [35] and demonstrated that in vitro and in vivo biofilms contain both type IV pilin protein and double-stranded DNA [14]. Greiner and colleagues showed that in vitro grown biofilms contain sialylated LOS as a key constituent of biofilms [36], which was later confirmed during in vivo chinchilla middle ear experiments [22]. Additionally, it was also shown that while sialylation of LOS promotes establishment of biofilms [37], incorporation of phosphorylcholine into LOS affects biofilm formation by NTHi in 6
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Fig. 6. Staining of NTHi biofilms with soybean agglutinin (SBA). NTHi biofilms were grown on Calu-3 cells and biofilm matrix was stained with SBA. Co-localization (yellow) of Fi176 WT biofilm staining and SBA staining is apparent. Fi176ΔpilA biofilm formation was reduced compared to Fi176 WT. (A) host cell nuclei: blue, (B) bacterial biofilm by Fi176 WT: red, (C) bacterial biofilm by Fi176ΔpilA, (D) lectin staining: green, (E) merge of A, B & D (magnification, x40). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 7. Inhibition and disruption of NTHi biofilms by proteinase and DNase. (A) Biofilm disruption/dispersal assays. After biofilm development by strain Fi176 (24 h at 37 °C under 5% CO2), non-adherent bacterial cells were removed, enzymes at the indicated concentrations were added and incubation allowed for an additional 24 h at 37 °C under 5% CO2 prior to staining with CV to quantify biofilm formation. (B) Biofilm inhibition assays. NTHi was grown overnight at 37 °C under 5% CO2, diluted 1:100 and incubated statically in BHI with proteinase K, or DNAse I at the indicated concentrations for 24 h and stained with CV. (C) Comparison of biofilm formation capacity among clinical NTHi strains. *P < 0.05 and **P < 0.001 compared with the untreated control.
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SBA, suggesting that NTHi biofilm matrix exopolysaccharide also contains GalNAc. In accordance with previous work by multiple groups [39–41], our results are also indicative for a role of PilA in NTHi biofilms grown on cell monolayers. In conclusion, we provide direct evidence that in vitro cultured airway epithelial cells support NTHi biofilm formation for prolonged infection time-points, and therefore provide valuable NTHi biofilm models for further studies of virulence and antimicrobial activity. Results from this study contribute to an improved understanding of NTHi biofilms in terms of biofilm architecture and constituents, and concomitant changes in host cell physiology in physiologically relevant models of infection.
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(2006), https://doi.org/10.1016/ j.diagmicrobio.2006.04.012. [20] M. Domenech, E. Pedrero-Vega, A. Prieto, E. García, Evidence of the presence of nucleic acids and β-glucan in the matrix of non-typeable Haemophilus influenzae in vitro biofilms, Sci. Rep. (2016), https://doi.org/10.1038/srep36424. [21] C. Cho, A. Chande, L. Gakhar, L.O. Bakaletz, J.A. Jurcisek, M. Ketterer, J. Shao, K. Gotoh, E. Foster, J. Hunt, E. O'Brien, M.A. Apicella, Role of the nuclease of nontypeable Haemophilus influenzae in dispersal of organisms from biofilms, Infect. Immun. (2015), https://doi.org/10.1128/IAI.02601-14. [22] J. Jurcisek, L. Greiner, H. Watanabe, A. Zaleski, M.A. Apicella, L.O. Bakaletz, Role of sialic acid and complex carbohydrate biosynthesis in biofilm formation by nontypeable Haemophilus influenzae in the chinchilla middle ear, Infect. Immun. (2005), https://doi.org/10.1128/IAI.73.6.3210-3218.2005. [23] J.D. Langereis, P.W.M. Hermans, Novel concepts in nontypeable Haemophilus influenzae biofilm formation, FEMS Microbiol. Lett. (2013), https://doi.org/10. 1111/1574-6968.12203. [24] T.D. Starner, N. Zhang, G.H. Kim, M.A. Apicella, P.B. McCray, Haemophilus influenzae forms biofilms on airway epithelia: implications in cystic fibrosis, Am. J. Respir. Crit. Care Med. (2006), https://doi.org/10.1164/rccm.200509-1459OC. [25] S. West-Barnette, A. Rockel, W.E. Swords, Biofilm growth increases phosphorylcholine content and decreases potency of nontypeable Haemophilus influenzae endotoxins, Infect. Immun. (2006), https://doi.org/10.1128/IAI.74.3.1828-1836. 2006. [26] B. Baddal, A. Muzzi, S. Censini, R.A. Calogero, G. Torricelli, S. Guidotti, A.R. Taddei, A. Covacci, M. Pizza, R. Rappuoli, M. Soriani, A. Pezzicoli, Erratum for Baddal et al., Dual RNA-seq of Nontypeable Haemophilus influenzae and Host Cell Transcriptomes Reveals Novel Insights into Host-Pathogen Cross Talk, mBio (2016), https://doi.org/10.1128/mbio.00373-16. [27] P.M. Power, S.D. Bentley, J. Parkhill, E.R. Moxon, D.W. Hood, Investigations into genome diversity of Haemophilus influenzae using whole genome sequencing of clinical isolates and laboratory transformants, BMC Microbiol. (2012), https://doi. org/10.1186/1471-2180-12-273. [28] S.J. Barenkamp, E. Leininger, Cloning, expression, and DNA sequence analysis of genes encoding nontypeable Haemophilus influenzae high-molecular-weight surface-exposed proteins related to filamentous hemagglutinin of Bordetella pertussis, Infect. Immun. (1992). [29] G. Ercoli, C. Tani, A. Pezzicoli, I. Vacca, M. Martinelli, S. Pecetta, R. Petracca, R. Rappuoli, M. Pizza, N. Norais, M. Soriani, B. Aricò, Lytm proteins play a crucial role in cell separation, outer membrane composition, and pathogenesis in nontypeable Haemophilus influenzae, mBio (2015), https://doi.org/10.1128/mBio. 02575-14. [30] M. De Chiara, D. Hood, A. Muzzi, D.J. Pickard, T. Perkins, M. Pizza, G. Dougan, R. Rappuoli, E.R. Moxon, M. Soriani, C. Donati, Genome sequencing of disease and carriage isolates of nontypeable Haemophilus influenzae identifies discrete population structure, Proc. Natl. Acad. Sci. U.S.A. (2014), https://doi.org/10.1073/pnas. 1403353111. [31] C.P. Ahearn, M.C. Gallo, T.F. Murphy, Insights on persistent airway infection by non-typeable Haemophilus influenzae in chronic obstructive pulmonary disease, Pathog. Dis. (2017), https://doi.org/10.1093/femspd/ftx042. [32] F.K. Raffel, B.R. Szelestey, W.L. Beatty, K.M. Mason, The Haemophilus influenzae Sap transporter mediates bacterium-epithelial cell homeostasis, Infect. Immun. (2013), https://doi.org/10.1128/IAI.00942-12. [33] M.R. Ketterer, J.Q. Shao, D.B. Hornick, B. Buscher, V.K. Bandi, M.A. Apicella, Infection of primary human bronchial epithelial cells by Haemophilus influenzae: macropinocytosis as a mechanism of airway epithelial cell entry, Infect. Immun. (1999). [34] D. Ren, K.L. Nelson, P.N. Uchakin, A.L. Smith, X.X. Gu, D.A. Daines, Characterization of extended co-culture of non-typeable Haemophilus influenzae with primary human respiratory tissues, Exp. Biol. Med. (2012), https://doi.org/10.
Funding This work was supported by the European Community’s Seventh Framework Programme EIMID ITN (European Institute of Microbiology and Infectious Diseases Initial Training Network, FP7-PEOPLE-2010264388). CRediT authorship contribution statement Buket Baddal: Conceptualization, Investigation, Data curation.
Methodology,
Validation,
Acknowledgements Parts of this work was conducted at Novartis Vaccines and Diagnostics, Siena, Italy. The authors would like to thank all members of the Research Center for the valuable discussions and contributions. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.micpath.2020.103985. References [1] L.O. Bakaletz, Immunopathogenesis of polymicrobial otitis media, J. Leukoc. Biol. (2010), https://doi.org/10.1189/jlb.0709518. [2] T.F. Murphy, Respiratory infections caused by non-typeable Haemophilus influenzae, Curr. Opin. Infect. Dis. (2003), https://doi.org/10.1097/00001432200304000-00009. [3] M. Ulanova, R.S.W. Tsang, Invasive Haemophilus influenzae disease: changing epidemiology and host-parasite interactions in the 21st century, Infect. Genet. Evol. (2009), https://doi.org/10.1016/j.meegid.2009.03.001. [4] T.F. Murphy, Vaccines for nontypeable Haemophilus influenzae: the future is now, Clin. Vaccine Immunol. (2015), https://doi.org/10.1128/CVI.00089-15. [5] M. Ikeda, N. Enomoto, D. Hashimoto, T. Fujisawa, N. Inui, Y. Nakamura, T. Suda, T. Nagata, Nontypeable Haemophilus influenzae exploits the interaction between protein-E and vitronectin for the adherence and invasion to bronchial epithelial cells, BMC Microbiol. (2015), https://doi.org/10.1186/s12866-015-0600-8. [6] I.L. Ahrén, H. Janson, A. Forsgren, K. Riesbeck, Protein D expression promotes the adherence and internalization of non-typeable Haemophilus influenzae into human monocytic cells, Microb. Pathog. (2001), https://doi.org/10.1006/mpat.2001. 0456. [7] E. Ronander, M. Brant, E. Eriksson, M. Mörgelin, O. Hallgren, G. Westergren‐Thorsson, A. Forsgren, K. Riesbeck, Nontypeable Haemophilus influenzae adhesin protein E: characterization and biological activity, J. Infect. Dis. (2009), https://doi.org/10.1086/596211. [8] V. Avadhanula, C.A. Rodriguez, G.C. Ulett, L.O. Bakaletz, E.E. Adderson, Nontypeable Haemophilus influenzae adheres to intercellular adhesion molecule 1 (ICAM-1) on respiratory epithelial cells and upregulates ICAM-1 expression, Infect. Immun. (2006), https://doi.org/10.1128/IAI.74.2.830-838.2006. [9] B. Euba, J. Moleres, C. Viadas, I.R. De Los Mozos, J. Valle, J.A. Bengoechea, J. Garmendia, Relative contribution of P5 and Hap surface proteins to nontypable Haemophilus influenzae interplay with the host upper and lower airways, PLoS One (2015), https://doi.org/10.1371/journal.pone.0123154. [10] B.L. Duell, Y.C. Su, K. Riesbeck, Host–pathogen interactions of nontypeable Haemophilus influenzae: from commensal to pathogen, FEBS Lett. (2016), https:// doi.org/10.1002/1873-3468.12351. [11] S.D. Reid, W. Hong, K.E. Dew, D.R. Winn, B. Pang, J. Watt, D.T. Glover, S.K. Hollingshead, W.E. Swords, Streptococcus pneumoniae forms surface‐attached communities in the middle ear of experimentally infected chinchillas, J. Infect. Dis.
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nontypeable haemophilus influenzae does not correlate with the level of biofilm formation in vitro, Infect. Immun. (2014), https://doi.org/10.1128/IAI.01445-13. [39] J.A. Jurcisek, J.E. Bookwalter, B.D. Baker, S. Fernandez, L.A. Novotny, R.S. Munson, L.O. Bakaletz, The PilA protein of non-typeable Haemophilus influenzae plays a role in biofilm formation, adherence to epithelial cells and colonization of the mammalian upper respiratory tract, Mol. Microbiol. (2007), https:// doi.org/10.1111/j.1365-2958.2007.05864.x. [40] E.M. Mokrzan, M.O. Ward, L.O. Bakaletz, Type IV pilus expression is upregulated in nontypeable Haemophilus influenzae biofilms formed at the temperature of the human nasopharynx, J. Bacteriol. (2016), https://doi.org/10.1128/JB.01022-15. [41] L.A. Novotny, J.A. Jurcisek, M.O. Ward, Z.B. Jordan, S.D. Goodman, L.O. Bakaletz, Antibodies against the majority subunit of type IV pili disperse nontypeable Haemophilus influenzae biofilms in a LuxS-dependent manner and confer therapeutic resolution of experimental otitis media, Mol. Microbiol. (2015), https://doi. org/10.1111/mmi.12934.
1258/ebm.2012.011377. [35] T.F. Murphy, C. Kirkham, Biofilm formation by nontypeable Haemophilus influenzae: strain variability, outer membrane antigen expression and role of pili, BMC Microbiol. (2002), https://doi.org/10.1186/1471-2180-2-7. [36] L.L. Greiner, H. Watanabe, N.J. Phillips, J. Shao, A. Morgan, A. Zaleski, B.W. Gibson, M.A. Apicella, Nontypeable Haemophilus influenzae strain 2019 produces a biofilm containing N-acetylneuraminic acid that may mimic sialylated O-linked glycans, Infect. Immun. (2004), https://doi.org/10.1128/IAI.72.7.42494260.2004. [37] W.E. Swords, M.L. Moore, L. Godzicki, G. Bukofzer, M.J. Mitten, J. VonCannon, Sialylation of lipooligosaccharides promotes biofilm formation by nontypeable Haemophilus influenzae, Infect. Immun. (2004), https://doi.org/10.1128/IAI.72.1. 106-113.2004. [38] C. Puig, S. Marti, P.W.M. Hermans, M.I. de Jonge, C. Ardanuy, J. Liñares, J.D. Langereis, Incorporation of phosphorylcholine into the lipooligosaccharide of
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Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Characterization of biofilm formation and induction of apoptotic DNA fragmentation by nontypeable Haemophilus influenzae on polarized human airway epithelial cells
T
Buket Baddala,b,∗ a b
Department of Medical Microbiology and Clinical Microbiology, Faculty of Medicine, Near East University, 99138, Nicosia, Cyprus Microbial Pathogenesis Research Group, DESAM Institute, Near East University, Nicosia, Cyprus
A R T I C LE I N FO
A B S T R A C T
Keywords: NTHi Biofilm formation Calu-3 cells Primary bronchial epithelium Apoptosis
Nontypeable Haemophilus influenzae (NTHi) is a common airway commensal and opportunistic pathogen that persists within biofilm communities in vivo. Biofilm studies so far are mainly based on assays on plastic surfaces. The aim of this work was to investigate the capacity of clinical NTHi strains to form biofilm structures on polarized Calu-3 human airway epithelial cells and primary normal human bronchial epithelial cells and to characterize the biofilm architecture. Formation of adherent NTHi biofilms post colonization of host cells at multiple time-points was evaluated using confocal laser scanning microscopy and electron microscopy. NTHi biofilms were analyzed in terms of biofilm height and presence of extracellular matrix components, and their apoptotic effects on epithelial cells were measured by TUNEL assay. Strain Fi176 was observed to form robust biofilms on airway epithelia over time, while disrupting the integrity of Calu-3 monolayer by 72 h of co-culture. NTHi biofilms were observed to induce apoptotic DNA fragmentation in host cells at 24 h post infection. Biofilm formation on cell monolayers by Fi176ΔpilA strain was markedly reduced compared to WT strain. Biofilm inhibition and disruption assays by crystal violet staining indicated that DNA and proteins are part of NTHi biofilms in vitro. Our findings highlight critical stages of NTHi pathogenesis following host colonization and provide useful biofilm models for future antimicrobial drug discovery investigations.
1. Introduction Nontypeable Haemophilus influenzae (NTHi) is a Gram-negative, unencapsulated opportunistic pathogen that commonly colonizes the human nasopharynx. While colonization can be asymptomatic, NTHi also establishes a pathogenic lifestyle within the host respiratory mucosa when host immune defenses are compromised or when bacterial virulence is enhanced [1]. NTHi is responsible for a wide range of respiratory tract infections including sinusitis, acute/chronic/recurrent otitis media (OM) and community-acquired pneumonia, and is implicated in majority of chronic infections of the lower respiratory tract such as cystic fibrosis (CF) and exacerbations of chronic obstructive pulmonary disease (COPD) [2]. The emergence of NTHi as a potential pathogen has been primarily observed after the introduction of H. influenzae serotype b (Hib) conjugate vaccine [3]. The high genetic diversity among strains is one of the main hurdles for the development of effective vaccines against NTHi, while the need for further efforts to understand the pathogenicity mechanisms, in order to identify novel
∗
antigens with cross-protective capacity, remains a high priority [4]. A critical first step in NTHi infections is adherence to host airway epithelial cells, which has been characterized in a variety of in vitro and in vivo experimental models [5–9]. Interactions of bacteria with host cells including adhesion, invasion, intracellular replication, intracellular trafficking, biofilm formation, bacterial metabolic adaptation, changes in stress response and evasion of host immunity have been the focus of NTHi research [10]. Similar to multiple other pathogenic bacteria affecting the respiratory tract such as Streptococcus pneumoniae [11], Pseudomonas aeruginosa [12] and Moraxella catarrhalis [13], NTHi infections are also complicated by the ability of the pathogen to rapidly establish biofilms, particularly in the middle ear [14–16]. Formation of biofilm communities is considered a virulence strategy due to their inherent antibiotic-resistant nature as well as their capacity to evade host defense factors [17]. Indeed, NTHi biofilms have recently been shown to be central to the pathogenesis of pulmonary infections such as bronchitis, CF and COPD [18]. Recent in vitro studies have confirmed that NTHi biofilms exhibit high antibiotic resistance [19].
Department of Medical Microbiology and Clinical Microbiology, Faculty of Medicine, Near East University, Nicosia, 99138, Cyprus E-mail address: [email protected].
https://doi.org/10.1016/j.micpath.2020.103985 Received 23 September 2019; Received in revised form 14 January 2020; Accepted 15 January 2020 Available online 20 January 2020 0882-4010/ © 2020 Elsevier Ltd. All rights reserved.
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scanning electron microscopy (JEOL JSM 6010LA InTouchScope).
Biofilm formation by the bacterial pathogen have been investigated in diverse in vitro static [20] as well as continuous-flow biofilm systems [21] and animal models of OM [22]. Using these systems, roles for lipooligosaccharides (LOS) sialylation, presence of phosphorylcholine, bacterial and eukaryotic DNA have been proposed to play a role in the development of biofilms [23]. More recently, transformed cell lines have been utilized to provide a more close-to-in vivo models of human airways, where bacterial biofilms have been grown on immortalized epithelial cell monolayers [24,25]. The use of physiologically relevant models of infection is vital to our understanding of infectious processes by pathogens. The present study assessed the capacity of clinical NTHi strains to form biofilms in vitro during prolonged infections on Calu-3 airway epithelial cells as well as primary bronchial epithelial cells with a pseudostratified epithelium phenotype, and investigated the characteristics of NTHi biofilms in terms of matrix components and changes in host cell physiology over time.
2.2. Bacterial culture and infection conditions NTHi strains Fi176 and Fi162 are clinical isolates obtained from patients with otitis media [27]. Strain R2846 was also isolated from the middle ear of a child with otitis media [28]. Deletion of pilA was performed to generate Fi176ΔpilA strain by allelic replacement of the entire coding sequence with an erythromycin resistance as described previously [29]. The genomic sequence of NTHi wild-type strain 176 was obtained using whole-genome sequencing [30]. All NTHi strains were reconstituted from frozen glycerol stock cultures, propagated on PoliVitex chocolate agar (bioMérieux) and incubated overnight at 37 °C with 5% CO2. For infection studies, NTHi was grown in brain heart infusion (BHI) broth (Difco Laboratories) supplemented with 10 μg/ml each of hemin (Fluka Biochemika) and nicotinamide-adenine nucleotide (NAD) (Sigma-Aldrich) until the optical density at 600 nm (OD600) reached 0.5. Bacteria were pelleted and resuspended in infection medium comprised of unsupplemented BEBM (for NHBE cells) and DMEM/F12 without penicillin/streptomycin, supplemented with %2 FBS (for Calu-3 cells) to prepare the inoculum. For infections, the inoculum was added to the apical surface of the cultures at multiplicity of infection (MOI) of 100:1, and incubated for 1 h at 37 °C and 5% CO2. Medium-only controls were used. After 1 h incubation, the inoculum was removed, and the apical surface was gently rinsed three times with 500 μl PBS in order remove any nonadherent bacteria. All infections were undertaken in triplicates. For biofilm formation studies, infected cultures were incubated for indicated periods of time and antibiotic-free basolateral medium was replaced every two days.
2. Materials and methods 2.1. Cell culture Calu-3 cells (ATCC No. HTB-55) were expanded in 75-cm2 flasks using DMEM/F12 supplemented with 1% MEM non-essential amino acid solution (Gibco), %10 Fetal Bovine Serum (FBS, Gibco) and 1% penicillin/streptomycin (Gibco). Cells were dissociated with %0.25 Trypsin-EDTA (Gibco) and seeded on semipermeable polyester membrane transwell supports (12-mm diameter, 0.4-μm pore size; Costar) at a cell density of 5 × 105 cells/ml. Medium (0.5 ml) was added to the basolateral chamber. When confluence was reached (day 4), medium on the apical surface was removed to produce air-liquid interface (ALI). Medium in the basolateral chamber was replaced every other day. Cells were cultured for an additional 10 days and transepithelial resistance (TEER) measurements were taken using an epithelial volt ohmmeter (EVOM2; World Precision Instruments) to ensure cell polarization. Transwell inserts with TEER values > 1000 Ω/cm2 were used for experimentation. To prepare cultures for infection, media with antibiotics were removed and cells were apically and basolaterally washed with phosphate-buffered saline (PBS, Sigma-Aldrich). After three washes with PBS, 0.5 ml antibiotic-free culture medium was added to basolateral chamber and cells were incubated with antibiotic free media for 24 h. For primary cell cultures, primary normal human bronchial epithelial (NHBE) cells (Clonetics-BioWhittaker, San Diego, CA) were used as previously described [26]. Briefly, cells were expanded in 75-cm2 flasks, using bronchial epithelial basal growth medium (BEBM; Lonza) supplemented with the BEGM (bronchial epithelial cell growth medium) BulletKit (Lonza) as recommended by the supplier at 37 °C in 5% CO2 until ~80% confluence, and used between passages 1 and 3. Cells were dissociated using StemPro Accutase cell dissociation reagent (Gibco) and were seeded onto semipermeable membrane supports (12mm diameter, 0.4-μm pore size; Costar) that were previously coated with a solution of collagen type I from rat tail (Gibco) at a concentration of 0.03 mg/ml. The cells were seeded at a density of 105 cells per well using bronchial air-liquid interface (B-ALI) medium (Lonza) supplemented with the B-ALI BulletKit (Lonza). When confluence was reached, the apical medium was removed and ALI was established to trigger differentiation. Cells were maintained at ALI for at least 28 days prior to use in biofilm assays, with the basolateral medium being changed every second day, to ensure a differentiated cell population. The apical side was rinsed with PBS every week to remove excess mucus production. Cell polarity and tight junction (TJ) barrier function were verified by TEER measurements using an epithelial volt ohmmeter. Cultures with TEER values of 800 Ω/cm2 were used for experimentation. Mucus secretion and cilia development were monitored by immunofluorescence microscopy (Zeiss LSM 710) and confirmed by
2.3. Immunofluorescence staining and confocal microscopy For each infection time-point, biofilm-containing transwell inserts were fixed with 4% paraformaldehyde. Membranes were permeabilized and blocked with 3% bovine serum albumin and 0.1% Triton X-100 in PBS. Incubation with rabbit anti-total NTHi serum (in-house), 1:5000, biotinylated soybean agglutinin (SBA, Vector Laboratories), 1:500, Streptavidin Alexa Fluor 594 Conjugate (Invitrogen), 1:1000, Alexa Fluor 647 phalloidin (Invitrogen), 1:1000 and Hoechst 33342 (Invitrogen), 1:10000 were performed either overnight at 4 °C or for 2 h at room temperature (RT). Samples were mounted using ProLong Gold Antifade Mountant (Invitrogen) and analyzed by confocal microscopy using a Zeiss LSM 710 confocal microscope. For z-stack imaging, at each time point, three stacked images at x100 magnification were obtained. Each of the stacked images were selected from an apical surface area at random. Three-dimensional rendering was performed using Imaris software (Bit-Plane, Inc.). Biofilm height on airway epithelia was measured using Zen 2.3 Image Analysis Software 3D Rendering Module. For apoptosis assays, cells were stained using ClickiT™ TUNEL Alexa Fluor™ 488 Imaging Assay (Invitrogen) according to manufacturer's instructions. Uninfected cells were used as negative controls and DNase I treated cells were employed as positive controls.
2.4. Electron microscopy All samples were fixed in 2.5% glutaraldehyde and 2.5% paraformaldehyde in 0.1 M sodium cacodylate buffer overnight, washed in buffer, and secondarily fixed in 1% osmium tetroxide in cacodylate buffer for 1 h. Samples were washed in water and block stained with 1% uranyl acetate for 1 h. After an hour, samples were dehydrated with ethanol using progressively increasing concentrations and dried by the critical point method using CO2 in a Balzers Union CPD 020 (BAL-TEC AG), sputter coated with gold in a Balzers MED 010 unit, and observed with a JEOL JSM 6010LA InTouchScope scanning electron microscope. 2
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orthogonal image sections in Fig. 2C. As morphological evidence by confocal microscopy suggested biofilm structures forming on cell monolayers, scanning electron microscopy (SEM) was employed to provide a more direct visualization of the biofilm matrix. Uninfected cells are shown in Fig. 3A. SEM imaging at 1 hpi revealed attachment of bacteria to apical surface of cells (Fig. 3B), while bacteria were observed to be actively dividing and forming biofilm structures by 24 h (Fig. 3C). At 72 hpi, NTHi biofilms were observed over airway microvilli, in which bacterial cells were enmeshed with exopolysaccharide matrix (white arrow) of the biofilm and host cell mucus as evident in Fig. 3D.
2.5. Biofilm formation assay on plastic For biofilm formation assays, all bacterial strains from overnight cultures were diluted 1:100 and incubated statically in BHI on plastic 24-well plates at 37 °C with 5% CO2. Uninoculated BHI only controls were used. After 24 h, the wells were gently washed once with 1 ml sterile PBS and allowed to dry for 10 min. Bacteria were stained with 1 ml of filter-sterilized 0.2% crystal violet (Sigma) and incubated for 30 min at RT. Crystal violet was removed from the wells, followed by two washes with 1 ml PBS. The dye was extracted by adding 1 ml of 96% ethanol to each well and incubating for 30 min at RT. The absorbance was measured at 540 nm using a Tecan Infinite 200 PRO plate reader. Inhibition of biofilm formation was assessed by adding 0.1 mg/ ml proteinase K or 2 U/mL DNase to bacteria in wells at the beginning of the incubation in the plates and quantified as above. For biofilm disruption assays, biofilms were grown on 24-well plates for 24 h, nonattached cells were removed, and then biofilms were subjected to treatment with 0.1 mg/ml proteinase K or 2 U/mL DNase. Cultures were further incubated for 24 h with the enzymes and were subjected to staining with crystal violet. Absorbance measurements were taken as described above.
3.2. Biofilm formation on primary normal human bronchial epithelial (NHBE) cells In order to confirm observations obtained with Calu-3 cell model, a fully differentiated airway epithelial model comprising of primary NHBE cells was used. When grown at ALI and provided necessary growth supplements, NHBE cells form a pseudostratified epithelium with TJs and adherence junctions (AJs), and contain a high population of ciliated cells (Fig. S1). The airway epithelium also exhibits TEER, mucus secretion and mucociliary activity, and therefore represents an environment similar to in vivo airway lumen. Differentiated NHBE cultures were infected apically with strain Fi176 and biofilm formation capacity at ALI was monitored in a time-course of infection by confocal microscopy. NTHi (red) were labelled with anti-total NTHi serum, bronchial epithelium was stained with phalloidin (green) and nuclei were counterstained with Hoechst 33342 (blue). As biofilm structures started being apparent at 24 hpi on Calu-3 cells, NHBE cells were fixed initially at this time-point and analyzed. At 24 hpi, bacterial aggregates (red) were observed mostly in between NHBE cell boundaries (green) on the apical side with a measured thickness of 4 μm (Fig. 4A). A progressive increase in biofilm mass forming on airway epithelia was observed at 48 and 72 hpi, in which biofilm thickness was 8 μm and 14 μm, respectively (Fig. 4B and C).
2.6. Statistical analysis All data are expressed as mean standard deviation (SD) of the triplicate experimental data. A two-tailed Student's t-test was used to determine the differences in biofilm formation between the control and each group. P value of < 0.05 was taken as significant. 3. Results 3.1. Biofilm formation on Calu-3 airway cells To investigate the capacity of NTHi to form biofilm on airway cells, Calu-3 lung adenocarcinoma cell line in a transwell system was used. Calu-3 cells were cultured on porous membrane at ALI to trigger cell polarization and formation of TJs (Fig. 1). Polarized monolayers were synchronically infected with NTHi strain Fi176 and monolayers were analyzed by immunofluorescent staining and confocal microscopy at 1 h, 24 h and 72 h post infection (hpi). Bacteria (red) were labelled with anti-total NTHi serum and epithelium was stained with phalloidin (gray) to visualize F-actin filaments. At 1 h post inoculation, adherent NTHi were found in low numbers scattered over the cell surface on the apical side of the monolayer (Fig. 2A). After 24 h of incubation, bacteria were observed to adapt to the host epithelium and multiply in number, forming microcolonies on airway epithelia (Fig. 2B). Importantly, bacteria were not exposed to any media apically in this model, mimicking the in vivo lung environment. No intracellular bacteria were detectable by microscopy. A drastic increase in the propensity of NTHi to form biofilms was evident at prolonged infection time-points, with aberrant rearrangements of the host cytoskeleton at 72 hpi as shown in
3.3. Induction of apoptosis in airway cells by NTHi biofilms The role of biofilm phenotype in apoptosis of infected Calu-3 cells was determined by terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) staining. TUNEL assay detects apoptotic cells that have undergone extensive DNA degradation during the late stages of apoptosis and is based on the ability of TdT to label blunt ends of double-stranded DNA breaks. Calu-3 cells infected with NTHi strain Fi176 at MOI 100:1 were analyzed at 1, 2, 4, 6, 24, 48 and 72 hpi by confocal laser scanning microscopy (CLSM) for the appearance of apoptotic cells. While apoptotic cells were not observed until 6 hpi (Fig. 5A–D), CLSM imaging showed that biofilms formed by strain Fi176 induced apoptosis in host cells at 24 h of infection, after which the number of TUNEL-positive cells increased exponentially at 48 and 72 hpi (Fig. 5E–G). Mock infected cells were used as negative controls
Fig. 1. Transwell model of Calu-3 airway epithelium and NTHi time-course infection. Calu-3 cells were cultured on 12 mm transwell permeable inserts at air-liquid interface to establish polarized monolayers. Polarized cell layers were infected with NTHi at a multiplicity of infection 100:1 and biofilm formation was monitored at 1, 24 and 72 hpi. 3
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Fig. 2. NTHi progressively forms biofilms on Calu-3 airway epithelial cell monolayers. Representative en face and orthogonal sections imaged by confocal laser scanning microscopy are shown (magnification, x100). (A) Few bacterial cells (red) attached on the apical surface of cells (gray) at 1 hpi. (B) At 24 hpi, NTHi forms microcolonies on host cell surface. (C) At 72 hpi, NTHi forms biofilms on airway epithelia and induces aberrant changes in host cell architecture. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) Fig. 3. Scanning electron microscopy of NTHi biofilms grown on Calu-3 monolayers. (A) Uninfected Calu-3 monolayers with microvilli 14 days post culture at air-liquid interface. (B) Bacterial cells attaching to host cell surface clearly visible at 1 hpi. (C) NTHi microcolonies and biofilm structures forming on cells at 24 hpi. (D) Thick biofilms observed over host cell microvilli with apparent biofilm exopolysaccharide (white arrows). Magnification shown for each section separately.
plates were employed. Treatment of preformed bacterial biofilms with 0.1 mg/ml proteinase K and 2 U/mL DNase separately caused significant dispersal of NTHi biofilms when compared to untreated wells (Fig. 7A), indicating that their matrix contains proteins and DNA important to their maintenance. Similarly, in biofilm inhibition assays, where bacteria were inoculated and incubated with the enzymes for 24 h, a significant reduction in biofilm-forming capacity of bacteria was observed (Fig. 7B). These data suggest that extracellular DNA and proteins are part of the NTHi biofilm matrix.
where no apoptosis was observed, whereas DNase I treated cells served as positive control for TUNEL assay in which enzyme treated host cells exhibited an apoptotic population. 3.4. Composition of NTHi biofilm matrix To be able to gain insights into the composition of NTHi biofilm matrix forming on Calu-3 airway epithelia, SBA was used to detect the presence of matrix exopolysaccharides in a more physiological model compared to microtiter assays, which are commonly performed on plastic surfaces. SBA is a lectin known to preferentially bind to oligosaccharide structures with terminal α- or β-linked N-acetylgalactosamine. In this assay, epithelial cell nuclei were labelled with Hoechst 33342 (Fig. 6A) and bacteria were labelled with anti-total NTHi serum (Fig. 6B). Immunofluorescence imaging of NTHi biofilms at 72 hpi revealed positive labeling by SBA (Fig. 6D), which indicates that NTHi biofilm matrix consists of sugar moieties such as oligosaccharide structures with terminal N-acetylgalactosamine as shown by the co-localization (yellow) of SBA staining (green) and bacterial biofilm (red) (Fig. 6E). For further analysis of the biofilm matrix, biofilm inhibition and disruption assays were set up for strain Fi176 using DNase I and proteinase K. As the treatment of host cells with these proteolytic and DNAdegrading enzymes would compromise the viability of eukaryotic cells, standard biofilm assays with crystal violet staining on plastic 12-well
3.5. Biofilm formation capacity of clinical NTHi strains A number of factors have been described to contribute to biofilm formation by bacterial pathogens. To be able to confirm microscopy results with a quantitative in vitro system, crystal violet assays have been performed. All three clinical isolates were compared for their biofilm forming capacity on plastic plates for 24 h. Crystal violet staining, a well-accepted bioassay, confirmed that strain Fi176 forms biofilms on plastic as it does on epithelial monolayers. Importantly, strain Fi176 formed significantly more biofilm biomass than strains Fi162 and R2846 (Fig. 7C). To be able to test the contribution of NTHi type IV pilus, in particular the major protein subunit PilA, to biofilm formation, strain Fi176 and its isogenic mutant strain Fi176ΔpilA were compared by crystal violet assays. A statistically significant difference between the biomass formed by strain Fi176 WT and Fi176ΔpilA was 4
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Fig. 4. NTHi forms thick biofilms on differentiated primary normal human bronchial epithelium. Confocal microscopy images of en face and orthogonal sections are shown (magnification, x40). (A) At 24 hpi, NTHi (red) forms microcolonies on NHBE cells (F-actin: green, nuclei: blue) with a height of 4 μm. (B) NTHi rapidly grows on airway epithelia and forms biofilm structures up to a height of 8 μm by 48 hpi. (C) Progressive increase in biofilm mass with a height of 14 μm is observed by 72 hpi. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
mainly adhesion, invasion and host evasion have been thoroughly studied using primary cell models of infection, limited data exists for biofilm formation on airway epithelia. In one of the first reports on NTHi biofilm formation on epithelial cells, NTHi strain 2019, a clinical strain from a COPD patient, was used and biofilm structures were observed on Calu-3 airway cells cultured on transwell inserts up to 5 days [24]. In a study by West-Barnette et al., immortalized human HMEEC-1 middle ear epithelial cells and 16HBE14o− airway epithelial cells were cultured at ALI on permeable membrane inserts, and electron microscopy revealed biofilm formation on apical surface by day 2 by NTHi strain 2019 [25]. In our study, clinical OM strain Fi176 formed significant biofilms on Calu-3 airway cells up to 3 days in co-culture. In accordance with a previous study which demonstrated that NTHI strain 86-028NP, a pediatric OM isolate, established biofilms on confluent monolayers of chinchilla middle ear epithelial cells and induced epithelial cell membrane ruffling and compromise of the epithelial cell membrane integrity [32], our results indicated that when NTHi biofilms form on airway cells at 24 h, the epithelial architecture loses integrity and cells undergo apoptosis as apparent by DNA fragmentation. The number of apoptotic host cells increases consistently up to 72 h of infection. Primary ciliated epithelial cells infected with NTHi have been previously characterized in terms of cell invasion and intracellular survival [33,34], however to our knowledge this is the first study to demonstrate NTHi biofilm forming on differentiated primary NHBE cells up to 72 h and with a biofilm height of 14 μm. The literature on NTHi biofilms have so far described some of the important factors which play a role in biofilm formation as well as the constituents of the biofilm matrix using in vitro microtiter or chamber slide, and in vivo chinchilla model of infection. Work from several
observed, confirming the role of PilA in NTHi biofilms (Fig. 7C). Importantly, these observations were confirmed by CLSM imaging of biofilms formed on Fi176ΔpilA infected Calu-3 monolayers, which in comparison to biofilms formed by WT strain were markedly reduced (Fig. 6C).
4. Discussion NTHi is a commensal bacterial pathogen most commonly associated with asymptomatic carriage, but under predisposing conditions develops strategies to cause persistent infections of the respiratory tract. There is a well-documented contribution of biofilm formation by NTHi to the chronic nature of OM, CF and COPD in the bronchial airway or middle ear [18,31]. Although a plethora of in vitro studies have been performed for elucidation of the role of biofilms in disease and their functional characteristics, majority of the work has been based on measurements of biofilm mass on plastic microtiter plates coupled with crystal violet staining. While these assays are highly informative when comparing a large number of clinical isolates for their biofilm forming capacity and are relatively low cost, they fail to provide physiologicallyrelevant conditions such as those found in the microenvironment of human airways. In this study, we have developed biofilm models for clinical NTHi strains grown on Calu-3 airway epithelial cells and on more physiologically-relevant in vitro differentiated primary human bronchial epithelium, and show that NTHi forms biofilms on airway epithelia over time with oligosaccharide structures with terminal α- or β-linked N-acetylgalactosamine (GalNAc), extracellular DNA and proteins as part of biofilm matrix. Although the interactions of NTHi with human epithelial cells, 5
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Fig. 5. NTHi biofilms induce apoptosis in host cells. Calu-3 cells were infected with NTHi strain Fi176 for 72 h and cells were analyzed by TUNEL assay. Bacteria are labelled in red, host cell nuclei in blue and TUNEL-positive cells in green. (A–D) No apoptotic cells were observed at 1–6 hpi. (E–F) NTHi induced apoptotic DNA fragmentation in host cells at 24 hpi. Apoptotic cells increased in number by 72 hpi (magnification, x10). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
a strain-specific manner [38]. In our study, biofilm inhibition and dispersal assays using standard crystal violet staining have shown that NTHi biofilms contain DNA and proteins, which is consistent with previous findings. In their study, Domenech et al. reported positive labeling for NTHi biofilm exopolysaccharides with only concanavalin A lectin, with α-Man and α/β-Glc specificity, in NTHi biofilms grown in glass-bottomed dishes [20]. In contrast to these findings, our results show positive staining of biofilms grown on Calu-3 monolayers with
groups have characterized the proteome of NTHi in biofilms [35] and demonstrated that in vitro and in vivo biofilms contain both type IV pilin protein and double-stranded DNA [14]. Greiner and colleagues showed that in vitro grown biofilms contain sialylated LOS as a key constituent of biofilms [36], which was later confirmed during in vivo chinchilla middle ear experiments [22]. Additionally, it was also shown that while sialylation of LOS promotes establishment of biofilms [37], incorporation of phosphorylcholine into LOS affects biofilm formation by NTHi in 6
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Fig. 6. Staining of NTHi biofilms with soybean agglutinin (SBA). NTHi biofilms were grown on Calu-3 cells and biofilm matrix was stained with SBA. Co-localization (yellow) of Fi176 WT biofilm staining and SBA staining is apparent. Fi176ΔpilA biofilm formation was reduced compared to Fi176 WT. (A) host cell nuclei: blue, (B) bacterial biofilm by Fi176 WT: red, (C) bacterial biofilm by Fi176ΔpilA, (D) lectin staining: green, (E) merge of A, B & D (magnification, x40). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 7. Inhibition and disruption of NTHi biofilms by proteinase and DNase. (A) Biofilm disruption/dispersal assays. After biofilm development by strain Fi176 (24 h at 37 °C under 5% CO2), non-adherent bacterial cells were removed, enzymes at the indicated concentrations were added and incubation allowed for an additional 24 h at 37 °C under 5% CO2 prior to staining with CV to quantify biofilm formation. (B) Biofilm inhibition assays. NTHi was grown overnight at 37 °C under 5% CO2, diluted 1:100 and incubated statically in BHI with proteinase K, or DNAse I at the indicated concentrations for 24 h and stained with CV. (C) Comparison of biofilm formation capacity among clinical NTHi strains. *P < 0.05 and **P < 0.001 compared with the untreated control.
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SBA, suggesting that NTHi biofilm matrix exopolysaccharide also contains GalNAc. In accordance with previous work by multiple groups [39–41], our results are also indicative for a role of PilA in NTHi biofilms grown on cell monolayers. In conclusion, we provide direct evidence that in vitro cultured airway epithelial cells support NTHi biofilm formation for prolonged infection time-points, and therefore provide valuable NTHi biofilm models for further studies of virulence and antimicrobial activity. Results from this study contribute to an improved understanding of NTHi biofilms in terms of biofilm architecture and constituents, and concomitant changes in host cell physiology in physiologically relevant models of infection.
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Funding This work was supported by the European Community’s Seventh Framework Programme EIMID ITN (European Institute of Microbiology and Infectious Diseases Initial Training Network, FP7-PEOPLE-2010264388). CRediT authorship contribution statement Buket Baddal: Conceptualization, Investigation, Data curation.
Methodology,
Validation,
Acknowledgements Parts of this work was conducted at Novartis Vaccines and Diagnostics, Siena, Italy. The authors would like to thank all members of the Research Center for the valuable discussions and contributions. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.micpath.2020.103985. References [1] L.O. Bakaletz, Immunopathogenesis of polymicrobial otitis media, J. Leukoc. Biol. (2010), https://doi.org/10.1189/jlb.0709518. [2] T.F. Murphy, Respiratory infections caused by non-typeable Haemophilus influenzae, Curr. Opin. Infect. Dis. (2003), https://doi.org/10.1097/00001432200304000-00009. [3] M. Ulanova, R.S.W. Tsang, Invasive Haemophilus influenzae disease: changing epidemiology and host-parasite interactions in the 21st century, Infect. Genet. Evol. (2009), https://doi.org/10.1016/j.meegid.2009.03.001. [4] T.F. Murphy, Vaccines for nontypeable Haemophilus influenzae: the future is now, Clin. Vaccine Immunol. (2015), https://doi.org/10.1128/CVI.00089-15. [5] M. Ikeda, N. Enomoto, D. Hashimoto, T. Fujisawa, N. Inui, Y. Nakamura, T. Suda, T. Nagata, Nontypeable Haemophilus influenzae exploits the interaction between protein-E and vitronectin for the adherence and invasion to bronchial epithelial cells, BMC Microbiol. (2015), https://doi.org/10.1186/s12866-015-0600-8. [6] I.L. Ahrén, H. Janson, A. Forsgren, K. Riesbeck, Protein D expression promotes the adherence and internalization of non-typeable Haemophilus influenzae into human monocytic cells, Microb. Pathog. (2001), https://doi.org/10.1006/mpat.2001. 0456. [7] E. Ronander, M. Brant, E. Eriksson, M. Mörgelin, O. Hallgren, G. Westergren‐Thorsson, A. Forsgren, K. Riesbeck, Nontypeable Haemophilus influenzae adhesin protein E: characterization and biological activity, J. Infect. Dis. (2009), https://doi.org/10.1086/596211. [8] V. Avadhanula, C.A. Rodriguez, G.C. Ulett, L.O. Bakaletz, E.E. Adderson, Nontypeable Haemophilus influenzae adheres to intercellular adhesion molecule 1 (ICAM-1) on respiratory epithelial cells and upregulates ICAM-1 expression, Infect. Immun. (2006), https://doi.org/10.1128/IAI.74.2.830-838.2006. [9] B. Euba, J. Moleres, C. Viadas, I.R. De Los Mozos, J. Valle, J.A. Bengoechea, J. Garmendia, Relative contribution of P5 and Hap surface proteins to nontypable Haemophilus influenzae interplay with the host upper and lower airways, PLoS One (2015), https://doi.org/10.1371/journal.pone.0123154. [10] B.L. Duell, Y.C. Su, K. Riesbeck, Host–pathogen interactions of nontypeable Haemophilus influenzae: from commensal to pathogen, FEBS Lett. (2016), https:// doi.org/10.1002/1873-3468.12351. [11] S.D. Reid, W. Hong, K.E. Dew, D.R. Winn, B. Pang, J. Watt, D.T. Glover, S.K. Hollingshead, W.E. Swords, Streptococcus pneumoniae forms surface‐attached communities in the middle ear of experimentally infected chinchillas, J. Infect. Dis.
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