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Infection and Immunity, July 2004, p. 4249-4260, Vol. 72, No. 7
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.7.4249-4260.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Nontypeable Haemophilus influenzae Strain 2019 Produces a Biofilm Containing N-Acetylneuraminic Acid That May Mimic Sialylated O-Linked Glycans

L. L. Greiner,1,{dagger} H. Watanabe,1,{dagger},{ddagger} N. J. Phillips,2,{dagger} J. Shao,1 A. Morgan,1 A. Zaleski,1 B. W. Gibson,3 and M. A. Apicella1*

Department of Microbiology, University of Iowa, Iowa City, Iowa 52242,1 Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446,2 Buck Institute for Age Research, Novato, California 949453

Received 26 November 2003/ Returned for modification 11 February 2004/ Accepted 19 March 2004


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ABSTRACT
 
Previous studies suggested that nontypeable Haemophilus influenzae (NTHI) can form biofilms during human and chinchilla middle ear infections. Microscopic analysis of a 5-day biofilm of NTHI strain 2019 grown in a continuous-flow chamber revealed that the biofilm had a diffuse matrix interlaced with multiple water channels. Our studies showed that biofilm production was significantly decreased when a chemically defined medium lacking N-acetylneuraminic acid (sialic acid) was used. Based on these observations, we examined mutations in seven NTHI strain 2019 genes involved in carbohydrate and lipooligosaccharide biosynthesis. NTHI strain 2019 with mutations in the genes encoding CMP-N-acetylneuraminic acid synthetase (siaB), one of the three NTHI sialyltransferases (siaA), and the undecaprenyl-phosphate {alpha}-N-acetylglucosaminyltransferase homolog (wecA) produced significantly smaller amounts of biofilm. NTHI strain 2019 with mutations in genes encoding phosphoglucomutase (pgm), UDP-galactose-4-epimerase, and two other NTHI sialyltransferases (lic3A and lsgB) produced biofilms that were equivalent to or larger than the biofilms produced by the parent strain. The biofilm formed by the NTHI strain 2019pgm mutant was studied with Maackia amurensis fluorescein isothiocyanate (FITC)-conjugated and Sambucus nigra tetramethyl rhodamine isocyanate (TRITC)-conjugated lectins. S. nigra TRITC-conjugated lectin bound to this biofilm, while M. amurensis FITC-conjugated lectin did not. S. nigra TRITC-conjugated lectin binding was inhibited by incubation with {alpha}2,6-neuraminyllactose and by pretreatment of the biofilm with Vibrio cholerae neuraminidase. Matrix-assisted laser desorption ionization—time of flight mass spectometry analysis of lipooligosaccharides isolated from a biofilm, the planktonic phase, and plate-grown organisms showed that the levels of most sialylated glycoforms were two- to fourfold greater when the lipooligosaccharide was derived from planktonic or biofilm organisms. Our data indicate that NTHI strain 2019 produces a biofilm containing {alpha}2,6-linked sialic acid and that the sialic acid content of the lipooligosaccharides increases concomitant with the transition of organisms to a biofilm form.


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INTRODUCTION
 
Bacterial biofilms have been defined as communities of bacteria that are intimately associated with each other and are in an exopolymer matrix. These biological units exhibit their own properties, which are quite different from the properties of the individual species in planktonic form (31). Numerous bacterial species are capable of producing biofilms.

Nontypeable Haemophilus influenzae (NTHI) is a gram-negative coccobacillus that frequently colonizes the human nasopharynx. NTHI is a frequent cause of otitis media in children (2) and acute bronchitis and pneumonia in patients with chronic obstructive pulmonary disease (35). Studies with a chinchilla otitis media model with NTHI infection have indicated that biofilms are produced as a part of this infection (10). Ehrlich et al. obtained scanning electron microscopy (SEM) and confocal images of biofilm formation on tympanostomy tubes collected from children with otitis media (10).

The purpose of this study was to identify NTHI genes involved in biofilm matrix biosynthesis. Recent studies by Swords and coworkers have shown that sialic acid (N-acetylneuraminic acid [Neu5Ac]) is a component of H. influenzae biofilms and that the CMP-Neu5Ac synthase must be functional (50). Using several different techniques to study biofilm formation, we found that an organism with a mutation in an NTHI gene homologous to the gene encoding an undecaprenyl-phosphate {alpha}-N-acetyl-glucosaminyltransferase (wecA) produces little or no biofilm. In addition, mutations in a gene encoding an NTHI sialyltransferase (siaA) and in a gene encoding CMP-Neu5Ac synthetase (siaB) resulted in significantly reduced biofilm production. Lectin studies and enzymatic analysis suggested that sialic acid is a terminal sugar attached to an N-acetylhexosamine (probably N-acetylgalactosamine) with an {alpha}2,6 linkage. Our studies also suggested that the levels of a number of sialylated lipooligosaccharide (LOS) glycoforms increase during biofilm and planktonic growth.


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MATERIALS AND METHODS
 
Bacteria and culture conditions. The bacterial strains used in this study are described in Table 1. NTHI strain 2019 is a clinical isolate obtained from a patient with chronic obstructive pulmonary disease (5). This strain was reconstituted from a frozen stock culture and was propagated on brain heart infusion (BHI) agar (Difco, Detroit, Mich.) supplemented with 10 µg of hemin (Sigma Chemical Co., St. Louis, Mo.) per ml and 10 µg of NAD (Sigma) per ml at 37°C in the presence of 5% CO2.


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  		TABLE 1. Bacterial strains and vectors

Construction of green fluorescent protein-expressing NTHI. NTHI strains were transformed by using a modification of the method of Williams et al. (53) and with plasmid pGB2::cat (1) encoding green fluorescent protein.

Construction of NTHI strain 2019galE. Previous studies have shown that galactose-4-epimerase is encoded by the HI0351 gene in the Institute for Genomics database (11). By using the upstream primer 5'-GCTGGTTATATCGGTTCT-3' and the downstream primer 5'-GATCAGAATAGCAAGTCGC-3', an 882-bp fragment of DNA was amplified from NTHI strain 2019 chromosomal DNA and ligated into pCR2.1 (Invitrogen, Carlsbad, Calif.). This fragment was sequenced and was shown to contain almost the entire HI0351 (galE) gene. A unique Eco47RIII site at bp 737 was identified within galE. The gene was digested, and an erythromycin resistance cassette was cloned into this site. Restriction digestions confirmed the mutation. This resistance gene was originally described by Monod et al. (32). The plasmid containing the mutated galE gene was linearized and transformed into NTHI strain 2019 as previously described (22). The mutation was confirmed by PCR and Southern blot analyses of NTHI strain 2019galE chromosomal DNA.

Construction of a wecA::Specr deletion mutant. To construct a mutant with a deletion mutation in NTHI strain 2019 wecA (HI1716), an NTHI strain 2019 phagemid genomic library was screened with a wecA digitonine-labeled probe. Positive plaques were purified, and excision was performed. Plasmid pBK-CMVHIA2wecA was isolated, and sequencing verified the presence of HI1714, HI1715, wecA (HI1716), and 912 bp of the 3' end of HI1719 in a 3,449-bp insert. The gene order in strain 2019 is different from that in H. influenzae Rd KW20, and open reading frames HI1718 and HI1719 are not adjacent to HI1716. The fragment was moved into pGEM3Zf(+) (Promega, Madison, Wis.) at the SacI and SamI sites. The plasmid was restricted with BclI and NsiI. This removed 682 bp of sequence internal to wecA. A spectinomycin resistance (Specr) cassette was cloned into the BclI and NsiI sites. This resistance gene was described by LeBlanc and coworkers and was isolated from Enterococcus faecalis (24). DNA sequencing and diagnostic DNA restriction enzyme digestion were performed to verify this construct. The mutation was introduced into NTHI strain 2019 by linearizing the plasmid construct as described above. Transformants were plated onto supplemented BHI agar containing 25 µl of spectinomycin per ml. The deletion mutation in NTHI strain 2019wecA was confirmed by PCR and Southern blot analyses.

Construction of a nanA::Kanr insertion mutant. In order to study incorporation of Neu5Ac into biofilms, a mutation in the Haemophilus N-acetylneuraminate lyase gene, nanA (The Institute for Genome Research locus HI0142), was generated. The DNA sequence of H. influenzae Rd KW20 was used to construct two primers, 5'-CCTACGATATGAATAGGATCATTACG-3' and 5'-CAGTAGCTAACCCCAATACAAAAG-3'. PCR was then used to amplify a 2,612-bp fragment of DNA, which was cloned into pCR2.1-TOPO (Invitrogen). This fragment was removed by EcoRI digestion, inserted into EcoRI-digested pUC19, and sequenced to verify the presence of Haemophilus nanA. A kanamycin resistance cassette (Kanr) was then inserted into nanA by using TN::EZ<KAN2> (Epicentre, Madison, Wis.) according to the manufacturer's instructions. The kanamycin resistance gene was originally from Tn903 (Epicentre). The plasmid containing the mutated nanA::Kanr gene was linearized and used to transform NTHI strains 2019 and 2019pgm, as previously described (22). The presence of Kanr in nanA in both mutants (NTHI strains 2019nanA and 2019pgm::nanA) was confirmed by PCR and Southern blot analyses.

Biofilm production assay. Biofilms produced by NTHI strain 2019 and the NTHI strain 2019 mutants (Table 1) were analyzed by using a microtiter plate assay described by Murphy and Kirkham (36) and O'Toole and Kolter (38, 39). This assay is referred to as the O'Toole-Kolter assay below. An overnight BHI broth culture of each NTHI strain was diluted 1:200 in either fresh BHI broth or RPMI 1640 supplemented with 20 µM Neu5Ac. Portions (200 µl) of the suspensions (~1 x 107 bacteria) were inoculated into the outside wells of 96-well tissue culture plates (Nalgene Nunc International Co., Naperville, Ill.) in quadruplicate. The plates were incubated at 37°C in the presence of 5% CO2 for 24 h. Before biofilm quantitation, the optical density at 490 nm (OD490) was determined to assess bacterial growth. To quantify biofilm formation, 20 µl of crystal violet (Fisher Scientific, Pittsburgh, Pa.) was added to each well, and the plates were incubated at room temperature for 15 min. The plates were then washed vigorously with distilled water and air dried. Then 230 µl of 95% ethanol was added to each well, and the OD600 was determined. All strains were tested in quadruplicate, and the average biofilm formation for three different experiments was calculated.

[14C]Neu5Ac incorporation studies. Studies were performed to compare [14C]Neu5Ac incorporation into NTHI strain 2019nanA and 2019pgm::nanA biofilms. The strains were grown in 96-well microtiter plates for 24 h at 37°C in the presence of 5% CO2 in RPMI medium (Gibco, Grand Island, N.Y.) supplemented with 0.5 µg of protoporphyrin IX per ml, 10 µg of NAD per ml, 20 µM Neu5Ac, and 82.5 nCi of [14C]Neu5Ac (55 mCi/mmol; American Radiolabeled Chemicals, Inc., St. Louis, Mo.) per ml. The medium was carefully removed from each well, and the wells were washed three times with distilled water. Each biofilm was harvested with 100 µl of Microscint (Packard, Meridian, Conn.), and 30 µl from each well was counted by using a TopCounter (Parchard, Downers Grove, Ill.).

Biofilm growth in a continuous-flow chamber. Strain 2019 and selected mutants were grown in a continuous-flow chamber identical to that described by Davies et al. (8). Morse's defined medium diluted 1:10, with or without 20 µM Neu5Ac, was used in these experiments (33). To inoculate the chamber, 1 ml of H. influenzae (density, 1 x 108 cells/ml) was placed in the chamber and incubated for 1 h. Then medium at a flow rate of 180 µl/min was added. For preparation of LOS for mass spectrometry analysis, a biofilm was allowed to form in the continuous-flow chamber for 5 days. During the final 6 h before the biofilm was harvested, the medium flowing over the biofilm was collected and centrifuged, and the resulting preparation was designated the planktonic bacteria. For mass spectrometry experiments, LOS extracted from this preparation was designated the planktonic LOS sample. The biofilm was harvested from the glass coverslip of the chamber, and LOS was extracted from this material. For mass spectrometry experiments, this sample was designated the biofilm LOS sample. The LOS isolated from bacteria grown under both of these conditions were compared to LOS from bacteria grown on defined media.

Laser scanning confocal microscopy of a continuous-flow chamber. To examine biofilm formation, green fluorescent protein-expressing NTHI strains were grown in a flow chamber (5 by 35 by 1 mm) whose size was similar to the sizes of flow chambers described previously (47). The biofilm medium was Morse's defined medium (34) diluted 1:10 with phosphate-buffered saline (PBS) and supplemented with 10 µg of hemin per ml, 10 µg of NAD per ml, and 1 µg of chloramphenicol (Sigma) per ml. In some experiments (see below), 20 µM Neu5Ac (Sigma) was added to this medium. To infect the flow chamber, approximately 108 CFU ml–1 in fresh biofilm medium was placed in the chamber and incubated at 37°C for 1 h. A biofilm medium flow was then started and maintained at a constant rate of 180 µl/min. For Neu5Ac-free experiments, protoporphyrin IX (0.5 µg/ml) was added to the medium instead of hemin. Confocal images were obtained with the Bio-Rad MRC-1024 laser scanning confocal viewing system. All the microscopes used in this study are located at the Central Microscopy Research Facility at the University of Iowa (Iowa City).

Viability staining of bacteria from continuous-flow chamber biofilms. To evaluate the viability of bacteria present within a biofilm matrix, NTHI strain 2019 was grown in a flow chamber as described above. After 2 or 5 days, the flow chamber was carefully disconnected. A LIVE/DEAD BacLight bacterial viability kit (Molecular Probes, Eugene, Oreg.) was used to visualize live and dead bacteria within the biofilm. Briefly, SYTO 9 (component A) and propidium iodide (component B) were mixed at a 1:1 ratio. Three microliters of the viability stain was added to 1 ml of PBS. The medium in the chamber was aseptically replaced with the stain-PBS mixture. The chamber was incubated for 15 min at 37°C. One milliliter of sterile PBS was then added to the chamber to flush away excess stain. Biofilm bacteria within the chamber were immediately visualized with a Zeiss 510 laser scanning confocal microscope at a magnification of x10. The resulting images were compiled as cross sections of a z series.

SEM analysis of the biofilm. Biofilms were processed for SEM (9) and were examined with an Hitachi S-4000 scanning electron microscope. Briefly, coverslips were fixed in a 2% osmium tetroxide-perfluorocarbon solution for 2 h, dehydrated with three 100% ethanol washes, and dried with a critical point dryer to preserve the biofilm structure. The processed coverslips were then mounted onto stubs by using colloidal silver and were sputter coated with gold palladium.

LOS preparation. LOS was prepared by a modification of the Hitchcock-Brown method (16). Organisms were grown on BHI agar or Morse's defined medium containing agar supplemented with 10 µg of hemin per ml, 10 µg of NAD per ml, and 20 µM Neu5Ac. Organisms isolated from a single plate were suspended in 2 ml of PBS to a final OD650 of 0.9. The bacteria were washed twice with PBS, resuspended in 200 µl of lysis buffer (0.06 M Tris, 10 mM EDTA, 2.0% sodium dodecyl sulfate; pH 6.8), and incubated in a boiling water bath for 5 to 10 min. The samples were allowed to cool, and 30 µl of a 2.5-mg/ml proteinase K (Sigma) solution was added to 150 µl of each boiled sample. The samples were then incubated at 37°C for 16 to 24 h. LOS was precipitated by adding 0.1 volume of 3 M sodium acetate and 2 volumes of 100% ethanol, placing the preparation on dry ice for 10 min or in a –80°C freezer for 1 h, and centrifuging it at 15,000 x g for 5 min. The LOS pellets were washed twice with 70% ethanol, the final volume of each preparation was adjusted to 180 µl with double-distilled H2O, and the preparations were lyophilized. For mass spectrometric analysis organisms in the planktonic and biofilm phases of a continuous-flow chamber after growth in Morse's defined medium were isolated and extracted as described above.

O deacylation of LOS samples. LOS (<100 µg) from planktonic or biofilm NTHI strain 2019 was O deacylated by treatment with 30 µl of anhydrous hydrazine (Sigma) at 37°C for 40 min with occasional vortexing. Samples were then cooled in an ice bath, treated with 5 volumes of ice-cold acetone added drop-wise, and allowed to sit at –20°C for 1 h. After centrifugation (12,000 x g, 30 min, 4°C), the supernatants were removed, and the pelleted O-deacylated LOS was washed with 100 µl of chilled acetone and centrifuged a second time (12,000 x g, 30 min, 4°C). Following removal of the supernatants, the pellets were dissolved in 100 µl of Milli-Q deionized water (Millipore, Corp., Billerica, Mass.) and centrifuged (12,000 x g, 30 min, 4°C) to remove traces of water-insoluble material remaining in the samples. Finally, the supernatants (water-soluble O-deacylated LOS) from this final centrifugation step were removed, transferred to new vessels, and evaporated to dryness. The LOS from plate-grown NTHI strain 2019 (0.5 mg) was O deacylated in a similar fashion by using 100 µl of anhydrous hydrazine.

Neuraminidase treatment of O-deacylated LOS. To remove Neu5Ac, aliquots of the O-deacylated LOS samples (<30 µg) were digested with immobilized neuraminidase from Clostridium perfringens type VI-A (Sigma) in 40 µl of 10 mM ammonium acetate (pH 6.0) for 21 h at 37°C. The immobilized enzyme was pelleted by centrifugation (12,000 x g, 20 min, 4°C), and the supernatants were removed. The pellets were washed twice with 50 µl of buffer, followed by centrifugation (12,000 x g, 20 min, 4°C). Combined supernatants were evaporated to dryness, redissolved in 50 µl of deionized water, and evaporated to dryness again.

MALDI-TOF mass spectrometry. The O-deacylated LOS samples were analyzed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry by using a Voyager-DE mass spectrometer (Applied Biosystems, Foster City, Calif.) equipped with a nitrogen laser (337 nm). All spectra were recorded in the negative-ion mode by using delayed extraction conditions as described in detail elsewhere (21). O-deacylated LOS was dissolved in 30 µl of deionized water, and 5-µl aliquots were desalted by drop dialysis on VSWP 0.025-µm-pore-size nitrocellulose membranes (Millipore Corp.) over deionized water for 80 min. The drops recovered were evaporated to dryness and then redissolved in 5 µl of deionized water. One-microliter aliquots of desalted O-deacylated LOS samples were then transferred into 0.5-ml microcentrifuge tubes containing a small amount of cation-exchange resin (Dowex 50W-X8, NH4+ form; Bio-Rad, Hercules, Calif.). Subsequently, 1-µl aliquots of a matrix solution (a saturated solution of 2,5-dihydroxybenzoic acid in acetone) were added to the samples. After brief mixing, 1-µl portions of the mixtures were delivered to a stainless steel MALDI target and allowed to air dry. Approximately 200 laser shots were acquired for each sample. The spectra were smoothed with a 19-point Savitsky-Golay function and mass calibrated with an external mass calibrant consisting of the renin substrate tetradecapeptide, insulin chain B (oxidized), and bovine insulin (all obtained from Sigma). For comparison purposes, a two-point correction was then done with the spectra by using the expected fragment ion for O-deacylated diphosphoryl lipid A (m/z 952.0) and the B3 glycoform of NTHI strain 2019 (m/z 2522.1). All masses were expressed as average mass values.

Lectin analysis of biofilms. Five-day biofilms produced by NTHI strain 2019pgm were subjected to lectin analysis. This strain was chosen because it cannot produce an acceptor for Neu5Ac on its LOS, and our studies had shown that it was capable of forming an amount of biofilm that was equal to or greater than the amount of biofilm formed by the parent strain. Biofilms were fixed in 4% paraformaldehyde and embedded in situ in OCT resin (Sakura Finetek USA, Inc., Torrance, Calif.) on the coverslip surfaces upon which they were formed. After hardening, each coverslip was removed by freezing the sample in liquid nitrogen and shattering the glass, which left the biofilm in the OCT resin. The biofilm was then cut into 1-µm-thick sections. These sections were studied by using fluorescent microscopy and the following lectins: Maackia amurensis fluorescein isothiocyanate (FITC)-conjugated lectin and Sambucus nigra tetramethyl rhodamine isocyanate (TRITC)-conjugated lectin (EY Laboratories, San Mateo, Calif.). In some experiments (see below), biofilms were incubated for 1 h at 37°C with 0.05 U of Vibrio cholerae neuraminidase (Sigma) per ml to remove Neu5Ac residues before lectin analysis. Binding inhibition experiments were performed by preincubating the S. nigra TRITC-conjugated lectin with 200 µg of {alpha}2,6-linked N-neuramyllactose per ml for 30 min.

Statistical analyses. Statistical analyses with the paired Student t test were performed by using Statview for Macintosh.


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RESULTS
 
NTHI biofilm formation. Previous studies with the chinchilla middle ear infection model and the microtiter plate biofilm assay suggested that NTHI can form biofilms (10). Our studies with NTHI strain 2019 in which the O'Toole-Kolter microtiter plate assay was used suggested that this strain was capable of forming a biofilm. The ability of strain 2019 to form a biofilm was confirmed in a continuous-flow chamber during 5 days of growth (Fig. 1A). A toluidine blue-stained frozen section of the NTHI strain 2019 biofilm embedded in OCT resin is shown in Fig. 1B. This figure shows that there was a tightly packed matrix, organisms at the bottom of the biofilm, and a more diffuse structure interlaced with water channels further from the slide surface. Higher-magnification SEM images are shown in Fig. 2. At the top of the structure, there was a pellicle that was formed by the biofilm matrix (Fig. 2A and B). Figure 2C shows a lateral view of water channels within the biofilm. There were fibrils, which may have been remnants of the biofilm matrix, extending between bacteria. Using the LIVE/DEAD stain, we were able to demonstrate that on day 2, viable organisms predominated throughout the biofilm, whereas dead organisms primarily localized to the glass slide surface on which the biofilm formed (Fig. 3A). In contrast, by day 5 the proportion of live organisms appeared to decrease, and dead organisms were present throughout the biofilm (Fig. 3B). This suggested that in the continuous-flow system, an NTHI biofilm may have a finite life span.



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  		FIG. 1. (A) Biofilm formation by NTHI strain 2019 after 5 days of growth in a continuous-flow chamber. (B) Toluidine blue-stained cryosection of an NTHI 5-day biofilm embedded in OCT resin. The bottom of the section is adjacent to the glass coverslip surface.



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  		FIG. 2. SEM micrographs of a 5-day NTHI biofilm. (A) Top surface of the biofilm. The surface cracked during the desiccation and dehydration processes, but the matrix of the biofilm surrounding organisms is shown. (B) Cross-sectional view of the biofilm. The coverslip surface upon which the biofilm formed is at the top. There are multiple water channels throughout the biofilm. (C) Higher magnification of the cross-sectional view. Fibrillous material connects the organisms within the biofilm.



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  		FIG. 3. Confocal microscopy of LIVE/DEAD-stained bacteria within biofilms on day 2 (A) and day 5 (B). The sections are vertical sections of a z series comprised of 10 5-µm optical sections. Biofilms were embedded in OCT resin and cryosectioned. Viable organisms (green) were present in each specimen; however, by day 5, a greater proportion of the organisms at the coverslip surface appeared to be nonviable (red). Scale bars = 100 µm.

Analysis of LOS glycoforms. It has been shown that bacterial gene expression within biofilms changes (52). To more precisely examine the expression of LOS glycoforms in NTHI strain 2019 during growth as a biofilm, LOS was isolated from biofilm, planktonic, and plate-grown bacteria. The isolated LOS was O deacylated by treatment with anhydrous hydrazine and analyzed by MALDI-TOF mass spectrometry. Previous data showed that NTHI strain 2019 produces a complex mixture of LOS glycoforms when it is grown in culture medium (12, 40). The major component of strain 2019 LOS contains a lactose moiety (Galß1->4Glcß1->) linked to HepI of the common core structure [HepIII{alpha}1,2->HepII{alpha}1,3->HepI{alpha}1,5->Kdo(P) (3-deoxy-D-manno-octulosonic acid phosphate)->lipid A] (see Table 2), which is characteristic of H. influenzae LOS (28, 40). In the present study, when strain 2019 was grown on solid medium supplemented with Neu5Ac, its LOS repertoire expanded to include new sialylated, disialylated, and polysialylated species (Fig. 4A and Table 2). The sialylated and disialylated forms of the major hexose (Hex)2 glycoforms, B3* and B3**, contained the sialyllactose moiety observed in other strains of H. influenzae (19, 26, 44). Asialo and sialylated glycoforms containing HexNAc (whose proposed compositions are consistent with structures found in other strains of H. influenzae) (41) were also more abundant in plate-grown strain 2019. Additionally, many of the higher-molecular-weight sialylated glycoforms observed are consistent with species found in plate-grown H. influenzae type b strain A2 (22).


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  		TABLE 2. LOS glycoforms observed in plate-grown, planktonic, and biofilm NTHI strain 2019



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  		FIG. 4. Mass spectrometry analyses of NTHI strain 2019 LOS isolated from a plate-grown day 5 biofilm and day 5 planktonic organisms. Sialylated glycoforms are indicated by red. For a complete description of the spectra see the text and Tables 2 and 3. A subscript paragraph sign indicates that the glycoform contains one phosphorylcholine moiety; the subscript numbers indicate the numbers of phosphoethanolamine moieties; and the number of superscript asterisks indicates the number of Neu5Ac moieties.

Compared to LOS obtained from plate-grown strain 2019, LOS obtained from both the planktonic and biofilm organisms showed increased heterogeneity (Fig. 4). One factor contributing to the increased heterogeneity was an overall shift to lower phosphorylation states, which resulted in a distribution of glycoforms containing one, two, or three phosphoethanolamines for each species. In addition to this trend, there was enhanced production of higher-molecular-weight and sialylated glycoforms in LOS obtained from planktonic and biofilm organisms compared to the production of these forms in LOS isolated from plate-grown strain 2019. The increases were more easily measured when all of the phosphorylation states for a given glycoform were combined, and the results for each sample were normalized (Table 3). When data were treated in this semiquantitative fashion, the MALDI results showed that there were increases in the amounts of C-H glycoforms in the LOS of planktonic and biofilm organisms. The higher-molecular-weight glycoforms were most abundant in the LOS derived from biofilm organisms, and many of these glycoforms were acceptors for sialic acid. Concomitantly, the overall levels of sialylated glycoforms in the LOS of planktonic and biofilm organisms were greater than the level in the LOS of plate-grown strain 2019 (Table 3). In a few cases, the levels of individual sialylated glycoforms were comparable under the three growth conditions. However, the levels of most sialylated glycoforms increased two- to fourfold when LOS was derived from planktonic or biofilm organisms. Such increases were observed for the doubly sialylated LOS glycoforms B**, D**, and E** of planktonic and biofilm organisms. While in most respects the LOS populations from planktonic and biofilm organisms appeared to be quite similar, the H* glycoform appeared to be expressed most abundantly in the LOS from biofilm organisms (Fig. 4C and Table 3).


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  		TABLE 3. Relative levels of asialo and sialylated LOS glycoforms in plate-grown, planktonic, and biofilm NTHI strain 2019

To confirm the assignments of the sialylated glycoforms, portions of the LOS samples isolated from bacteria grown under the three conditions were treated with immobilized neuraminidase. The MALDI spectrum of the neuraminidase-treated LOS sample from biofilm organisms is shown in Fig. 4D. In all three neuraminidase-treated LOS samples, peaks identified as sialylated glycoforms were shifted by the loss of one or more Neu5Ac residues.

Analysis of biofilm formation by NTHI mutants. In order to determine the role that specific carbohydrates might play in NTHI biofilm formation, we studied a group of NTHI strain 2019 mutants in which complex carbohydrate biosynthesis was impaired. Figure 5 shows the results of this study. Seven NTHI strain 2019 mutants were studied by using a microtiter biofilm (O'Toole-Kolter) assay. Interestingly, NTHI mutants 2019galE and 2019pgm formed biofilms. Our previous studies showed that the LOS of mutant 2019pgm was truncated at the triheptose core. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the mutant 2019galE LOS was also severely truncated and had none of the higher-molecular-mass glycoforms which are acceptors for sialylation (data not shown). This suggested that glucose, galactose, and possibly mannose were not components of the biofilm matrix. Three other mutants, 2019wecA, 2019siaB, and 2019siaA, exhibited significant reductions in biofilm formation when the O'Toole-Kolter assay was used. Strain 2019wecA had a mutation in a gene with high homology (e–101) to the gene encoding undecaprenyl-phosphate {alpha}-N-acetylglucosaminyltransferase of Escherichia coli K-12. Previous studies in our laboratory (unpublished data) and studies of Hood and coworkers (17) indicated that a mutation in this gene does not affect LOS biosynthesis. Data obtained from O'Toole-Kolter assays suggested that this transferase might be involved in the first step in biofilm matrix biosynthesis (that is, addition of an initial N-acetylhexosamine to the undecaprenol carrier lipid). Figures 6A and B show the results of a confocal analysis of biofilm formation by strains 2019gfp and 2019wecA::gfp, respectively, at day 5 in a continuous-flow chamber when defined medium was used. The data confirmed our O'Toole-Kolter assay results, since essentially no biofilm was formed by 2019wecA, while a 50- to 150-µm-thick biofilm was formed by NTHI strain 2019. We performed a similar study with strain 2019siaB that showed that there was a reduction in the thickness of the biofilm (20 to 30 µm) at day 5 (Fig. 6C). O'Toole-Kolter assays were also used to study three strain 2019 sialyltransferase mutants (strains with mutations in lsgB, lic3A, and siaA). A mutation in siaA resulted in a significant reduction in biofilm formation, while mutations in lsgB and lic3A did not (Fig. 5). O'Toole-Kolter and continuous-flow chamber studies suggested that Neu5Ac was an important component of the biofilm. To confirm this suggestion, we again studied biofilm formation using a continuous-flow chamber, but this time we used Neu5Ac-limiting conditions. A chemically defined medium supplemented with NAD and hemin and without or with 20 µM Neu5Ac was perfused through separate chambers inoculated with NTHI strain 2019gfp. These studies showed that there was reduced biofilm formation in the continuous-flow chamber perfused with medium without Neu5Ac (Fig. 6D and E). We obtained similar results when the O'Toole-Kolter assay was used (Fig. 7A). [14C]Neu5Ac uptake studies were performed to confirm that Neu5Ac was incorporated into the biofilm produced by NTHI strain 2019pgm. These studies were performed by using NTHI strains 2019nanA and 2019pgm::nanA. H. influenzae cannot synthesize sialic acid, but it can degrade sialic acid to N-acetylmannosamine through the action of N-acetylneuraminate lyase (NanA). Figure 7B shows that NTHI strain 2019pgm::nanA incorporated [14C]Neu5Ac into biofilms as efficiently as NTHI strain 2019nanA incorporated this compound into biofilms and LOS.



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  		FIG. 5. Results of O'Toole-Kolter assays obtained by using strain 2019 and seven strain 2019 mutants. Three mutants (siaA, siaB, and wecA mutants) produced significantly less biofilm than the parent strain produced. The other mutants produced amounts of biofilms that were similar to or greater than the amount produced by the parent strain.



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  		FIG. 6. Images obtained from laser scanning confocal microscopy examination of vertical cross sections of 5-day biofilms. The cross sections are comprised of sixty 1-µm optical sections. Continuous-flow chambers were inoculated with NTHI strain 2019gfp (A), with NTHI strain 2019wecA::gfp (B), with NTHI strain 2019siaB::gfp (C), or with NTHI strain 2019gfp in medium supplemented with 20 µg of Neu5Ac per ml (D) or in medium without Neu5Ac (E). Scale bar, 50 µm.



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  		FIG. 7. (A) O'Toole-Kolter assay results showing that there was a significant reduction (P < 0.005) in biofilm production when NTHI strain 2019 was grown in defined medium without Neu5Ac (striped bar) compared to the growth of organisms in the presence of Neu5Ac (solid bar). (B) Neu5Ac incorporation after 24 h of growth of NTHI strain 2019nanA (stippled bar) and NTHI strain 2019pgm::nanA (striped bar) in defined medium in the presence of [14C]Neu5Ac. [14C]Neu5Ac uptake in six control wells to which bacteria were not added is also indicated (solid bar). This study demonstrated that NTHI strain 2019pgm::nanA incorporated Neu5Ac into a carbohydrate structure. Since this mutant could not incorporate Neu5Ac into its LOS, the Neu5Ac was most likely incorporated into the biofilm. The error bars indicate one standard deviation. The values are the mean values calculated from data obtained from eight wells (A) or nine wells (B).

Analysis of biofilm composition by lectin-binding studies. To further confirm that Neu5Ac was a component of the biofilms, biofilms embedded in OCT resin were studied with M. amurensis and S. nigra lectins conjugated to FITC and Texas Red isothiocyanate (TRITC), respectively. M. amurensis lectin preferentially bound to a terminal Neu5Ac {alpha}2,3-Gal (48), as observed for H. influenzae LOS, and S. nigra lectin bound preferentially to terminal Neu5Ac {alpha}2,6-Gal (43). Lectin binding was compared by using biofilms produced by strains 2019 and 2019pgm. The latter strain produced a LOS that was severely truncated, and there were no acceptors for Neu5Ac on its LOS. Thus, any Neu5Ac detected in lectin-binding studies of the strain 2019pgm biofilm would have been present only in the biofilm matrix. Strain 2019 and 2019pgm biofilms were collected after 5 days of culture in a continuous-flow chamber and embedded in OCT resin. Figures 8A to C show the results of a microscopic analysis of the 5-day strain 2019 biofilm stained with M. amurensis and S. nigra lectins before and after treatment with sialidase. Both lectins bound to the biofilm but to different portions of the structure. The S. nigra TRITC-conjugated lectin bound diffusely throughout the biofilm. The M. amurensis FITC-conjugated lectin bound to punctate areas around and within the biofilm, suggesting that lectin binding occurred on NTHI strain 2019 LOS. After sialidase treatment, neither lectin bound to the biofilm. We observed a somewhat different pattern of lectin binding when we analyzed the biofilm produced by strain 2019pgm. The S. nigra TRITC-conjugated lectin bound diffusely to the strain 2019pgm biofilm, similar to what was observed for the strain 2019 biofilm. In addition, S. nigra lectin binding was prevented by pretreatment of the biofilm with sialidase (Fig. 9) or by preincubation of the S. nigra TRITC-conjugated lectin with {alpha}2,6-N-acetylneuramyllactose (data not shown). As Fig. 9 shows, there was no change in the binding of M. amurensis FITC-conjugated lectin to the biofilm after sialidase treatment. This suggested that the binding of this lectin was not to sialic acid and that M. amurensis FITC-conjugated lectin binding either was nonspecific or involved another component of the biofilm. These studies suggested that Neu5Ac is present in the strain 2019 and 2019pgm biofilms in an {alpha}2,6 linkage.



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  		FIG. 8. (A to C) Confocal micrographs of OCT resin-embedded, cryosectioned biofilms produced by NTHI strain 2019 that were labeled with S. nigra TRITC-conjugated lectin (A) and M. amurensis FITC-conjugated lectin (B). Panel C is a merged image of panels A and B. (D to F) Confocal micrographs of OCT resin-embedded, cryosectioned biofilm produced by NTHI strain 2019 labeled with S. nigra TRITC-conjugated lectin (D) and M. amurensis FITC-conjugated lectin (E) after a 1-h exposure to 0.05 U of V. cholerae neuraminidase. Panel F is a merged image of panels D and E.



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  		FIG. 9. (A to C) Confocal micrographs of OCT resin-embedded, cryosectioned biofilm produced by NTHI strain 2019pgm labeled with S. nigra TRITC-conjugated lectin (A) and M. amurensis FITC-conjugated lectin (B). Panel C is a merged image of panels A and B. (D to F) Confocal micrographs of OCT resin-embedded, cryosectioned biofilm produced by NTHI strain 2019pgm labeled with S. nigra TRITC-conjugated lectin (D) and M. amurensis FITC-conjugated lectin (E) after a 1-h exposure to 0.05 U of V. cholerae neuraminidase. Panel F is a merged image of panels D and E.


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DISCUSSION
 
Biofilms are complex communities of microorganisms that develop on surfaces in diverse environments (13). They are found in many different environments, including industrial pipelines, ventilation systems, catheters, and medical implants, and they are involved in disease in both humans and animals. As shown by studies of biofilms, these structures are dynamic and start by attachment of bacteria to a surface; this is followed by formation of microcolonies and, finally, development of the mature, structurally complex biofilms (13). Bacteria eventually detach from the mature biofilm and enter the surrounding fluid phase, becoming planktonic organisms that can initiate biofilm development on other parts of the surface.

The mechanisms involved in the initial attachment are different in different microorganisms. The initiation of a biofilm can occur in one of three ways. The first way is by redistribution of attached cells by surface motility. O'Toole and Kolter (38) demonstrated that the type IV pili of Pseudomonas aeruginosa play an important role in surface adherence. The second mechanism by which biofilm formation can occur is from the binary division of surface-attached bacterial cells (15). The third and final mechanism is recruitment of bacterial cells from the surrounding medium (51).

After the initial attachment, the cells must convert from reversible attachment to irreversible attachment, in which the cells switch from a weak interaction with the substratum to permanent bonding through extracellular polymers. In addition to formation of the exopolymers, the bacteria form channels and pores and redistribute away from the substratum (7).

The maintenance of a biofilm is attributed to the development and maintenance of the exopolysaccharide matrix (6). More than 300 proteins that are not detected in planktonic bacteria have been detected in bacteria from mature biofilms (43). These proteins fall into the following functional classes: metabolism, phospholipid and lipopolysaccharide biosynthesis, membrane transport and secretion, and adaptation and protective mechanisms. In addition, biofilm bacteria are considered to be in the stationary phase partially because of the accumulation of acylhomoserine lactone within clusters (48).

Detachment is a physiologically regulated event in which a bacterium releases from the biofilm and becomes a planktonic organism that moves on to attach to another surface. Many different mechanisms may contribute to the detachment process. O'Toole and Kolter (38) demonstrated that starvation may lead to detachment by an unknown mechanism. Streptococcus mutans produces a surface protein, releasing enzyme, that mediates the release of cells from biofilms (25). A possible trigger for release of this matrix-degrading enzyme could be cell density. In addition, the presence of homoserine lactones may cause biofilm reduction, as demonstrated with Rhodobacter sphaeroides (38, 42).

In P. aeruginosa, flagella and type IV pilus-mediated twitching motility play important roles in surface aggregation (38). In E. coli, flagella, type I pili, and curli fimbriae are implicated in biofilm formation (20). Motility is not absolutely necessary as many nonmotile bacteria, such as Staphylococcus epidermidis and S. mutans, can also form biofilms. Microorganisms within a biofilm undergo changes in gene expression compared to the gene expression in plate-grown or planktonic bacteria (52). It has been demonstrated that Pseudomonas putida undergoes phenotypic changes in protein expression such that different stages of biofilm development can be recognized (43).

Recent studies have provided evidence that H. influenzae can produce a biofilm during otitis media in humans and in the chinchilla middle ear during experimental otitis media. Murphy and Kirkham (36) demonstrated that H. influenzae pili may play a role during growth in the O'Toole-Kolter microtiter plate assay. Using two different systems (the O'Toole-Kolter assay and a continuous-flow system), we found that NTHI strain 2019 can form a biofilm. We used these systems to identify genes that are involved in the formation of the extracellular polymeric substances of the NTHI strain 2019 biofilm. These studies have shown that WecA, SiaB, and SiaA appear to be involved in the formation of NTHI strain 2019 extracellular polymeric substances.

The wecA gene of NTHI strain 2019 exhibits high homology to the same gene in E. coli, Yersinia pestis, and Salmonella enterica serovar Typhimurium (<e–100). Previous studies have indicated that WecA plays no role in H. influenzae LOS biosynthesis (17, 29, 30). This gene codes for undecaprenyl-phosphate {alpha}-N-acetylglucosaminyltransferase, homologs of which are involved in the initial step in enterobacterial common antigen (29, 30, 37) and O-antigen biosynthesis in Salmonella enterica serovar Borreze (23) and P. aeruginosa (4). S. mutans rgpG, which also exhibits homology to E. coli wecA, is involved in the biosynthesis of an extracellular polysaccharide (45). We demonstrated, using the O'Toole-Kolter assay and a continuous-flow chamber, that a biofilm is not produced by NTHI mutant 2019wecA::gfp. This suggests that NTHI wecA is involved in the initial step in biosynthesis of a biofilm and that the biofilm is synthesized on undecaprenol pyrophosphate.

NTHI SiaB is a CMP-Neu5Ac synthetase, and NTHI SiaA is a sialyltransferase (22). Mutation of either NTHI strain 2019siaB or NTHI strain 2019siaA resulted in significantly reduced biofilm production, as determined by the O'Toole-Kolter assay and in continuous-flow chambers. In contrast to NTHI strain 2019wecA cultures, in which no biofilm formed by 5 days, a small but detectable biofilm was present in a continuous-flow chamber inoculated with the NTHI 2019siaB and NTHI 2019siaA mutants. NTHI has two other sialyltransferases, Lic3A and LsgB (19, 22). However, a mutation in the gene encoding either of these sialyltransferases did not alter NTHI strain 2019 biofilm formation. When H. influenzae lsgB and lic3A are mutated, SiaA can sialylate H. influenzae LOS (18); however, the primary role of SiaA most probably is involvement in biofilm formation. Our studies demonstrated that NTHI mutant 2019pgm can produce a biofilm that is equivalent to or greater than the biofilm formed by the parent strain. This indicates that glucose and galactose are probably not components of the NTHI biofilm because pgm codes for an enzyme essential for the biosynthesis of nucleotide derivatives of these sugars. If the nucleotide versions of these sugars are not formed, they cannot be incorporated into the biofilm matrix. There are a limited number of known acceptors for Neu5Ac, and these acceptors are most commonly galactose, N-acetylgalactosamine, N-acetylglucosamine, or another sialic acid (14). The formation of a Neu5Ac-containing biofilm by strain 2019pgm recognized by the S. nigra lectin suggests that the acceptor for Neu5Ac in the biofilm is a hexosamine, either N-acetylgalactosamine or N-acetylglucosamine.

We studied the binding of M. amurensis and S. nigra lectins to NTHI strain 2019 and 2019pgm biofilms. Studies in which we used the S. nigra lectin, before and after V. cholerae neuraminidase treatment, provided further evidence that Neu5Ac is the terminal sugar in the biofilm. M. amurensis lectin binds preferentially to Neu5Ac in an {alpha}2,3 linkage (46). It can also bind to lactose. S. nigra lectin binds preferentially to Neu5Ac in an {alpha}2,6 linkage. This binding appears to be specific, and there is minimal inhibition by high concentrations of lactose. We analyzed the biofilm produced by NTHI mutant 2019pgm because this bacterium does not produce an LOS with an acceptor for sialylation, which allowed us to study Neu5Ac incorporation into the biofilm alone. Specific binding of the S. nigra lectin combined with the nonspecific binding of the M. amurensis lectin to an NTHI strain 2019pgm biofilm indicated that Neu5Ac is incorporated into the biofilm via an {alpha}2,6 linkage.

NTHI LOS becomes more heterogeneous as bacteria adapt from growth on plates to a biofilm or planktonic existence, with changes seen in LOS phosphorylation, sialylation, and size. Sialylated, disialylated, and polysialylated LOS species are more abundant. In general, the levels of most of the sialylated glycoforms were two- to fourfold greater in LOS isolated from biofilm or planktonic organisms. This was especially true of the double sialylated glycoforms. These findings may reflect a change in the mechanisms by which Neu5Ac is metabolized. NTHI cannot synthesize Neu5Ac and obtains this compound from its environment. Recent studies of a chinchilla middle ear infection suggested that Neu5Ac incorporation into LOS is necessary for pathogenicity (3). Increased sialylation of the LOS of bacteria within a biofilm or in the planktonic phase may result from increased Neu5Ac uptake or diversion of Neu5Ac into pathways that lead to its incorporation into other carbohydrate structures. These adaptations could enhance bacterial survival within the host environment.

It is intriguing to speculate that NTHI, an airway colonizer and pathogen, has adapted to this environment by producing a biofilm that has structures that mimic the sialylated O-linked glycans of mucin. Moreover, it is also worth considering the possibility that there may be a functional or structural association between a newly formed biofilm and the altered LOS glycoform population, which with biofilm formation are enriched in sialic acid-containing species. Future studies will be directed at analyzing the biofilm exopolysaccharide structure and examining changes in gene expression that modify the LOS phenotype of NTHI within a biofilm.


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ACKNOWLEDGMENTS
 
This work was supported by grants AI24616 and AI30040 (to M.A.A.) and grant AI31254 (to B.W.G.) from the Public Health Service, National Institute of Allergy and Infectious Diseases, National Institutes of Health.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, University of Iowa, 51 Newton Rd., Iowa City, IA 52242. Phone: (319) 335-7807. Fax: (319) 335-9006. E-mail: michael-apicella{at}uiowa.edu. Back

Editor: J. N. Weiser

{dagger} L.L.G., H.W., and N.J.P. contributed equally to work described in this paper. Back

{ddagger} Present address: Department of Internal Medicine, Institute of Tropical Medicine, Nagasaki University, Nagaski, Japan. Back


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Infection and Immunity, July 2004, p. 4249-4260, Vol. 72, No. 7
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.7.4249-4260.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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