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Infection and Immunity, March 2006, p. 1588-1596, Vol. 74, No. 3
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.3.1588-1596.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Biofilm Formation by Moraxella catarrhalis In Vitro: Roles of the UspA1 Adhesin and the Hag Hemagglutinin

Melanie M. Pearson, Cassie A. Laurence, Sarah E. Guinn, and Eric J. Hansen*

Department of Microbiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, Texas 75390-9048

Received 1 September 2004/ Returned for modification 19 October 2004/ Accepted 16 December 2005


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ABSTRACT
 
Mutant analysis was used to identify Moraxella catarrhalis gene products necessary for biofilm development in a crystal violet-based assay involving 24-well tissue culture plates. The wild-type M. catarrhalis strains that formed the most extensive biofilms in this system proved to be refractory to transposon mutagenesis, so an M. catarrhalis strain was constructed that was both able to form biofilms in vitro and amenable to transposon mutagenesis. Chromosomal DNA from the biofilm-positive strain O46E was used to transform the biofilm-negative strain O35E; transformants able to form biofilms were identified and subjected to transposon-mediated mutagenesis. Biofilm-negative mutants of these transformants were shown to have a transposon insertion in the uspA1 gene. Nucleotide sequence analysis revealed that the biofilm-positive transformant T14 contained a hybrid O46E-O35E uspA1 gene, with the N-terminal 155 amino acids being derived from the O46E UspA1 protein. Transformant T14 was also shown to be unable to express the Hag protein, which normally extends from the surface of the M. catarrhalis cell. Introduction of a wild-type O35E hag gene into T14 eliminated its ability to form a biofilm. When the hybrid O46E-O35E uspA1 gene from T14 was used to replace the uspA1 gene of O35E, this transformant strain did not form a biofilm. However, inactivation of the hag gene did allow biofilm formation by strain O35E expressing the hybrid O46E-O35E uspA1 gene product. The Hag protein was shown to have an inhibitory or negative effect on biofilm formation by these M. catarrhalis strains in the crystal violet-based assay.


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INTRODUCTION
 
Moraxella catarrhalis is an important cause of otitis media in infants and very young children (9, 21). In adults with chronic obstructive pulmonary disease, M. catarrhalis is known to be a significant cause of infectious exacerbations (28, 38). In addition, M. catarrhalis has been shown to be an infrequent cause of several other diseases including pneumonia and sinusitis (for a review, see reference 28).

The ability of this organism to colonize the mucosal surface of the nasopharynx is key to its ability to cause disease in other anatomic regions, because this colonization event provides a foothold for M. catarrhalis in its human host. In fact, nasopharyngeal colonization with M. catarrhalis is common throughout infancy, and a high rate of colonization with this organism is associated with an increased risk of otitis media (10). The mechanism(s) essential for colonization of the nasopharyngeal mucosa by M. catarrhalis has not been determined conclusively, although a number of M. catarrhalis gene products that may be involved in this process have been identified in the past few years. The UspA1 and UspA2H proteins have both been shown to function as adhesins for human epithelial cells in vitro (22); more recently, the Hag (MID) protein was shown to bind to both A549 human lung cells (11, 18) and primary cultures of human middle ear epithelial cells (18). The M. catarrhalis OmpCD protein has been shown to bind both middle ear mucin (36) and human lung cells in vitro (17), and a novel outer membrane protein (McaP) that exhibits both adhesin and lipolytic activities was recently shown to bind several different human cell lines in vitro (40). In addition, antibody to a surface-exposed epitope of M. catarrhalis lipooligosaccharide inhibited attachment of this organism to Chang conjunctival cells in vitro (19), and it was recently shown that M. catarrhalis can express type IV pili (25).

While several putative adhesins of M. catarrhalis have been described, their relative contributions to colonization of the nasopharynx remain to be determined. Similarly, nothing is known about whether these or other M. catarrhalis gene products may be involved in biofilm development. It is now recognized that biofilm formation by bacteria is relevant not only to environmental and industrial microbiology but also to infectious diseases (5, 7). First well studied with respect to oral microbiology (7, 41), there are now many examples of pathogens that form biofilms (7) and of how gene expression by a bacterium growing in a biofilm can differ from that of the same organism growing in the planktonic state (i.e., in broth) (7, 32, 37). With regard to gram-negative pathogens that cause otitis media, it was recently shown that nontypeable Haemophilus influenzae forms a biofilm both in vitro (29) and in the middle ear of experimentally infected chinchillas (8).

In the present study, we initially evaluated a large number of M. catarrhalis strains for their biofilm formation ability in a crystal violet-based assay and found that isolates of this pathogen vary widely in their ability to form biofilms in vitro. After using genetic transformation to introduce biofilm formation ability from a biofilm-forming strain into a non-biofilm-forming strain, we determined the identity of two genes (uspA1 and hag) whose encoded protein products are involved, in different ways, in biofilm formation in vitro by these two strains of M. catarrhalis.


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MATERIALS AND METHODS
 
Bacterial strains and culture conditions. The wild-type and mutant strains of M. catarrhalis used in this study are listed in Table 1. Additional strains of M. catarrhalis tested by the crystal violet-based assay included strains isolated in North Carolina, Tennessee, New York, Finland, and England. The M. catarrhalis O46E strain used in this study lacked the ability to express UspA2H because of apparent slipped-strand mispairing in the poly(A) tract located at the very start of the uspA2H open reading frame (ORF) (W. Wang, M. M. Pearson, A. S. Attia, R. J. Blick, and E. J. Hansen, manuscript in preparation). Expression of the UspA2H protein by O46E caused a hyperautoaggregation phenotype that rapidly removed these organisms from suspension; therefore, the UspA2H-negative phase variant was used in this study. This strain also possessed nine G residues in the poly(G) tract of its uspA1 gene and consequently expressed a relatively low level of UspA1 (23). M. catarrhalis strains were routinely grown in brain heart infusion (BHI) broth (Difco/Becton Dickinson, Sparks, MD) at 37°C with aeration or on BHI agar plates at 37°C in an atmosphere of 95% air-5% CO2. For some transformation experiments, Todd-Hewitt (TH) broth (Difco) solidified with 1.5% (wt/vol) Bacto agar (Difco) was used in place of BHI agar. Antibiotic supplementation for mutant selection involved the use of kanamycin (15 µg/ml), spectinomycin (15 µg/ml), chloramphenicol (0.6 µg/ml), or dihydrostreptomycin sulfate (750 µg/ml). M. catarrhalis biofilm formation assays were performed without antibiotics.


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  		TABLE 1. Bacterial strains and plasmids used in this study

MAbs and Western blot analysis. Western blot analysis using monoclonal antibody (MAb) 24B5, which is specific for the UspA1 protein, and MAb 5D2, which is reactive with the Hag protein, was performed as previously described (2, 22, 33).

Crystal violet-based biofilm assay. This method was adapted from the protocol published by O'Toole and Kolter (31) for use with Pseudomonas fluorescens. M. catarrhalis was inoculated into 5 ml BHI broth in polystyrene plastic tubes (each, 17 by 100 mm). These cultures were incubated at 37°C with aeration until the bacterial density reached at least 2 x 108 to 4 x 108 CFU/ml, corresponding to a reading of 125 Klett units, as determined by the use of a Klett-Summerson colorimeter (Klett Manufacturing Company, New York, NY). These cultures were diluted 1:100 in BHI, and 2-ml portions of this suspension were loaded, in triplicate, into a 24-well tissue culture plate (Corning Incorporated, Corning, NY), and incubated at 37°C for 19 h without aeration. The broth was then removed from each well and replaced by 2 ml of Medium 199 tissue culture medium containing Earle's balanced salt solution, L-glutamine, and HEPES (Fisher Scientific, Pittsburgh, PA) plus 100 µl of 0.7% (wt/vol) crystal violet (Sigma, St. Louis, Mo.). After 15 min at room temperature, the fluid contents of each well were decanted, and the well was washed three times with deionized water. A 2-ml volume of 95% ethanol was added to each well, and the plate was rocked gently for 15 min. A 1.5-ml portion of this ethanol solution was then transferred to a new 24-well plate, and the absorbance at 570 nm was measured using a SPECTRAFluor Plus fluorometer (Tecan, Research Triangle Park, NC).

PCR. PCR was accomplished with ExTaq DNA polymerase (PanVera, Madison, WI) according to the manufacturer's instructions. Chromosomal DNA templates were prepared from M. catarrhalis cells using the Easy-DNA kit (Invitrogen, Carlsbad, CA).

Nucleotide sequence analysis. PCR products were sequenced using an ABI PRISM 377 DNA sequencer (Applied Biosystems, Foster City, CA) and analyzed using the MacVector analysis package (version 6.5; Oxford Molecular Group, Campbell, CA).

Genetic transformation of M. catarrhalis. This method was adapted from that described by Juni (20). A 4-µl volume of M. catarrhalis chromosomal DNA (containing 1 µg DNA) was spotted onto the surface of a BHI agar plate. A toothpick was then used to transfer one or two M. catarrhalis colonies to the DNA spot. Using the same toothpick, the DNA and bacteria were spread over a circular area approximately 2 cm in diameter. Following incubation at 37°C with 95% air-5% CO2 for 6 h, the bacterial growth was suspended in 300 µl BHI broth in a 1.5-ml Eppendorf centrifuge tube, diluted in BHI broth, and spread onto BHI agar plates (containing an appropriate antimicrobial supplement as necessary).

Construction of biofilm-forming transformants. The biofilm-negative M. catarrhalis strain O35E was transformed with chromosomal DNA from the biofilm-positive M. catarrhalis strain O46E by the agar plate-based method described above. To enrich for biofilm-positive transformants, a 100-µl portion from the 300-µl suspension was added to a 24-well tissue culture plate containing 2 ml BHI broth and incubated overnight at 37°C. The broth was removed, and each well was washed once with BHI. Fresh BHI was added, and the plates were incubated for an additional 6 h at 37°C. After the broth was removed again, adherent bacteria were scraped from the walls of the wells, serially diluted, and spread onto BHI agar plates to obtain isolated colonies. Individual colonies were then grown as patches on BHI agar and used to inoculate 24-well tissue culture plates to measure biofilm formation in the crystal violet-based assay.

Transposon mutagenesis. M. catarrhalis transformant strains T13 and T14 were mutagenized with the EZ::TN<KAN-2> transposome (Epicenter, Madison, WI) according to the manufacturer's instructions. Briefly, M. catarrhalis was grown in BHI broth to a density of 108 CFU/ml. The bacteria were washed three times with 10% (vol/vol) glycerol and resuspended in a 1/100 volume of 10% glycerol. A 20-µl portion of these cells was electroporated with 1 µl of the transposome and then inoculated into 0.5 ml BHI broth. After 3 h of incubation at 37°C, the cells were plated on BHI agar supplemented with kanamycin. Kanamycin-resistant transformants were screened for loss of biofilm formation ability by transferring a swatch of bacterial growth from a BHI-kanamycin agar plate into a 24-well tissue culture plate containing 2 ml BHI broth per well and proceeding with the crystal violet-based assay.

Enrichment for allelic exchange by congression. A streptomycin-resistant mutant of M. catarrhalis strain ETSU-9 was obtained by spreading 109 to 1010 CFU of this strain onto BHI agar supplemented with streptomycin and incubating overnight. The development of spontaneous resistance to streptomycin is frequently associated with a point mutation in the rpsL gene (30). To determine whether a mutation in the M. catarrhalis rpsL gene had occurred, a 0.8-kb fragment containing the entire rpsL gene was amplified by PCR using the oligonucleotide primers 5'-GGAATTCACTCAAGTGAAAATACGGAAAATC-3' and 5'-GAGGTACCGACGTCTTGGCATAATAGTT-3', together with chromosomal DNA from the streptomycin-resistant mutant ETSU-9-Smr. Nucleotide sequence analysis confirmed that a single nucleotide change at residue 128 in the rpsL gene resulted in a single altered amino acid (i.e., K43R). A 3-kb fragment of DNA containing the mutated rpsL gene was generated by PCR with the primers 5'-TGGCGAAGAACTCAAGCAAACAGC-3' and 5'-ACGCCACCAACAGCACAATAAACC-3'. To replace the hag gene of transformant T14 with the hag gene from strain O35E, a 6.5-kb amplicon encompassing the O35E hag gene was amplified by PCR from O35E chromosomal DNA using the primers 5'-TTGCCCCATATCTGTACG-3' and 5'-GGTCATGGTGAAAGAGAATC-3'. A 5-µl portion of this PCR amplicon (approximately 0.5 µg DNA) was mixed with 0.5 µl of the 3-kb rpsL PCR amplicon (approximately 25 ng DNA) and was used to transform the spectinomycin-resistant hag mutant T14.HG. This latter mutant has a hag gene that was inactivated by insertion of a spectinomycin resistance cartridge derived from pELHGSPEC (Table 1). Transformants were selected on TH agar supplemented with streptomycin. Isolated colonies were then patched onto TH agar plates containing spectinomycin to identify spectinomycin-sensitive transformants in which the mutated hag gene had been replaced by all or part of the O35E wild-type hag gene. Nucleotide sequence analysis of the hag gene was used to confirm the occurrence of allelic exchange. The resultant transformants were designated T14.O35EHG1 through T14.O35EHG5.

Construction of M. catarrhalis mutants. Insertional mutagenesis of the uspA1 gene was accomplished by transformation of M. catarrhalis strains with pUSPA1KAN (2). Mutations in the hag gene were similarly constructed by transformation with pELHGSPEC (33). To introduce the hybrid O46E-O35E uspA1 gene from transformant T14 into strain O35E, the uspA1 gene from T14 was amplified by PCR and used to transform the uspA1 mutant O35E.1 (which has a kan cartridge inserted in its uspA1 gene) (1). Transformants were screened for loss of kanamycin resistance to identify those in which allelic exchange had occurred. The presence of the entire hybrid O46E-O35E uspA1 sequence in transformant O35E.14U1 was confirmed by nucleotide sequence analysis. To construct a mutant unable to express the corC gene product, this gene and its flanking regions were amplified by PCR using the primers 5'-ATTTATGATGAATTGCGACC-3' and 5'-TGGTGAGCAGTTTTTACCG-3' and chromosomal DNA from M. catarrhalis transformant T14 as the template. This fragment was ligated into pCR2.1 (Invitrogen) and the resultant plasmid, designated pMP14COR, was digested with SphI and NdeI to remove a fragment of approximately 200 nucleotides from the corC ORF. A promoterless cat cartridge (26) was ligated with the linearized pMP14COR plasmid. Escherichia coli strain DH5{alpha} was electroporated with this ligation reaction mixture; the plasmid from a resultant chloramphenicol-resistant transformant was designated pMP14CORcat. This plasmid containing the inactivated cor gene was used to transform M. catarrhalis transformant T14 to obtain the corC mutant T14.COR.

Bacterial attachment assays. Attachment of M. catarrhalis strains to Chang conjunctival epithelial cells was determined as described previously (22), except that the Chang monolayers were washed (to remove unattached bacteria) and harvested immediately after centrifugation. Adherence was calculated as the percentage of bacteria attached to the Chang cells relative to the initial inoculum.


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RESULTS
 
M. catarrhalis strains vary in their ability to form biofilms. Previous studies from our laboratory (33) and that of Struthers (3) have shown that at least two M. catarrhalis strains (i.e., O35E and ATCC 25238) can form biofilms on the porous cellulose filter in the Sorbarod continuous flow biofilm system. However, the Sorbarod model does not lend itself to screening large numbers of strains for biofilm formation. In contrast, the crystal violet-based assay for biofilm formation described by O'Toole and Kolter (31) is very useful for large-scale screens.

We modified this crystal violet-based assay to measure the ability of 51 M. catarrhalis strains to form biofilms in 24-well tissue culture plates. These isolates comprised both strains isolated from patients with M. catarrhalis disease and strains obtained from the nasopharynges of healthy individuals. The results of these assays indicated that M. catarrhalis strains have widely varying abilities to form biofilms, with most strains forming biofilms very poorly under these in vitro conditions (data not shown). However, strain O46E (Fig. 1A) readily formed biofilms in this assay and was initially selected for genetic analysis because this strain had previously been shown to be capable of undergoing genetic transformation involving cloned fragments of M. catarrhalis DNA (22).


Figure 1
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  		FIG. 1. Biofilm formation by parent and transformant strains of M. catarrhalis. (A) Photograph of biofilm formation in a 24-well tissue culture plate. M. catarrhalis O46E, O35E, and transformant T14 were grown in this 24-well plate; the wells were stained with crystal violet and then washed three times with deionized water. The arrows indicate biofilm growth on the plastic in two of these wells. (B) Quantitative measurement of biofilm formation by strains O46E, O35E, transformant T13, and transformant T14 by the crystal violet assay. The absorbance (A570) of the ethanol extract is plotted on the vertical axis. Each assay was performed in triplicate and repeated three times.

Transformation of biofilm formation ability into strain O35E. Initial efforts to identify the genes essential for biofilm formation by strain O46E involved the use of a transposon mutagenesis system which has been reported to be effective for mutagenizing M. catarrhalis strain O35E (18, 24). However, strain O46E was refractory to mutagenesis by this system, as were the other M. catarrhalis strains that readily formed biofilms by this system (data not shown). It should be noted here that strain O35E formed little or no biofilm in the crystal violet-based assay (Fig. 1A). This was in contrast to its demonstrated ability to form a biofilm in the Sorbarod system (33). However, the biofilm-negative phenotype of strain O35E in the crystal violet-based biofilm assay provided the opportunity to use O35E as a recipient in transformation experiments designed to introduce biofilm formation ability into a biofilm-negative strain. By using transformation to obtain a biofilm-positive strain which was also amenable to transposon mutagenesis, we sought to have an unbiased approach to the identification of gene products essential for biofilm formation by these strains of M. catarrhalis in the crystal violet-based assay in vitro.

Chromosomal DNA from the biofilm-positive strain O46E was used to transform the biofilm-negative strain O35E; enrichment for transformants able to form biofilms was accomplished as described in Materials and Methods. From 365 colonies tested, we obtained several biofilm-positive transformants. Two of these, designated T13 and T14 (Fig. 1B), were selected for further analysis. Biofilm formation by T14 is depicted in Fig. 1A. Subsequent comparison of the outer membrane protein and lipooligosaccharide profiles of these two transformants revealed that they were similar if not identical to each other and to strain O35E (data not shown). In addition, these two transformants had very similar growth rates in broth (data not shown).

Isolation of biofilm-negative transposon insertion mutants. Transformant strains T13 and T14 were subjected to transposon mutagenesis, and a total of 398 kanamycin-resistant colonies were obtained. The occurrence of random transposon insertions was confirmed by Southern blot analysis (data not shown). All of these putative insertion mutants were screened in the crystal violet-based assay, and three mutants deficient in biofilm formation ability were identified and designated T13/38, T13/54, and T14/27 (Fig. 2A). Initial attempts to use marker rescue cloning in E. coli (i.e., cloning of the transposon and flanking chromosomal DNA) to identify the gene(s) disrupted by the transposon in these mutants were not successful.


Figure 2
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  		FIG. 2. Biofilm formation by wild-type, mutant, and transformant strains of M. catarrhalis as measured by the crystal violet-based assay. (A) Transformant T13, transformant T14, wild-type strain O35E, transposon mutant T13/38 (transposon in uspA1), transposon mutant T13/54 (transposon in uspA1), transposon mutant T14/27 (transposon in corC), uspA1 mutant T13.1, uspA1 mutant T14.1, and corC mutant T14.COR. (B) O46E parent strain and its uspA1 mutant O46E.l-4.

Additional characterization of the biofilm-positive transformants T13 and T14 included assessment of their ability to attach to Chang conjunctival epithelial cells in vitro (1, 22) because it was possible that the ability to form biofilms might augment the ability of M. catarrhalis to attach to human cells. Both T13 and T14 exhibited an increased ability to attach to Chang cells relative to strain O35E (Fig. 3). Because the UspA1 protein has been shown to be essential for maximal attachment of M. catarrhalis strain O35E to Chang cells (22), the uspA1 gene was inactivated in both T13 and T14. Both the uspA1 mutants T13.1 and T14.1 exhibited a greatly reduced ability to attach to Chang cells in vitro (Fig. 3). Interestingly, these same uspA1 mutants were also much less able to form biofilms than their immediate parent strains (Fig. 2A). Inactivation of the uspA1 gene of the O46E parent strain also caused a reduction in biofilm formation (Fig. 2B).


Figure 3
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  		FIG. 3. Attachment of wild-type, transformant, and mutant strains of M. catarrhalis O35E to Chang conjunctival epithelial cells in vitro. The ability of these strains to attach to Chang cells was measured as described in Materials and Methods. Strains used in this experiment include wild-type O35E and its uspA1 mutant O35E.1, transformant T13 and its uspA1 mutant T13.1, and transformant T14 and its uspA1 mutant T14.1.

The fact that these uspA1 mutants of transformants T13 and T14 were biofilm deficient prompted us to investigate whether any of the three original kanamycin-resistant transposon mutants described above (i.e., T13/38, T13/54, and T14/27) might have had a transposon insertion in the uspA1 gene. PCR-based amplification of the uspA1 gene from transposon mutants T13/38 and T13/54 indicated that both of these mutants had transposon insertions in the uspA1 gene; this was confirmed by nucleotide sequence analysis of the amplicons. Mutant T14/27, however, did not have a transposon insertion in its uspA1 gene. (The transposon-disrupted gene in T14/27 is discussed in a later section.)

Transformants T13 and T14 possess a hybrid uspA1 gene. The fact that inactivation of the uspA1 gene in transformants T13 and T14 (Fig. 2A) and in the O46E parent strain (Fig. 2B) adversely affected biofilm formation by these strains raised the possibility that T13 and T14 both might express a hybrid UspA1 protein that contained one or more segments from the O46E UspA1 protein. This could have resulted from transformation and allelic exchange involving a fragment of O46E chromosomal DNA containing all or a portion of the uspA1 gene. Comparison of the uspA1 ORF from transformant T14 with the uspA1 ORFs from O46E and O35E revealed that T14 did indeed possess a hybrid uspA1 sequence, with the 5' portion of the ORF corresponding to that of the O46E uspA1 ORF and the remainder of the ORF matching O35E uspA1 sequence. This hybrid uspA1 gene encoded a predicted protein of 830 amino acids, with the first 155 amino acids being derived from strain O46E and the last 675 amino acids derived from strain O35E (Fig. 4). In addition, the poly(G) tract of this hybrid uspA1 gene contained 10 G residues instead of the 9 G residues present in the poly(G) tract of the O46E uspA1 gene (data not shown); this resulted in a higher level of UspA1 protein expression by T14 than by O46E (Fig. 5C). Phase variation of the poly(G) tract in the 5'-untranslated region of the uspA1 gene has been described previously (23). Like other UspA1 proteins characterized previously (4), these UspA1 proteins migrated more slowly by sodium dodecyl sulfate-polyacrylamide gel electrophoresis than would be predicted from their calculated masses. It should also be noted that, although the calculated mass of the mature wild-type UspA1 protein from strain O46E (88,468 Da) is greater than that of the mature O35E UspA1 protein (83,375 Da), the former protein migrated more rapidly than the O35E UspA1 protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. 5C). Nucleotide sequence analysis of the uspA1 gene from transformant T13 showed that this strain also had the same hybrid uspA1 sequence as that found in transformant T14 (data not shown), a finding which suggested that T13 and T14 likely were descendants of the same transformant. For this reason, only T14 was used in subsequent experiments.


Figure 4
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  		FIG. 4. Pileup comparison of the UspA1 proteins from the biofilm-positive strain O46E, the biofilm-positive transformant T14, and the biofilm-negative strain O35E. The black arrowhead indicates the crossover point between the O46E and O35E proteins that resulted in the T14 hybrid UspA1 protein.


Figure 5
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  		FIG. 5. Effect of Hag expression on biofilm formation by transformant T14. Western blot analysis of whole-cell lysates was accomplished using the Hag-specific MAb 5D2 (A) and the UspA1-specific MAb 24B5 (C) as the primary antibody probes. The number of G residues contained in the poly(G) tract in the hag ORF of each strain is listed in panel B. Biofilm formation was measured by means of the crystal violet-based assay (D). Strains used in these experiments include transformant T14, transposon mutant T14/27, corC mutant T14.COR, O46E, O35E, Hag-positive T14.O35EHG1, Hag-negative T14.O35EHG2, Hag-positive T14.O35EHG3, Hag-negative T14.O35EHG4, and Hag-negative T14.O35EHG5. Molecular mass position markers (in kilodaltons) are present on the left sides of panels A and C.

Characterization of mutant T14/27. To determine the location of the transposon insertion in the biofilm-negative mutant T14/27 (which did not have a transposon insertion in its uspA1 gene), a chromosomal EcoRV fragment containing the transposon was cloned into pACYC184. Analysis of this DNA insert revealed that the transposon had inserted into an ORF with homology to the E. coli corC gene, which encodes a protein suggested to be involved in magnesium efflux (13). However, when the wild-type M. catarrhalis O35E corC gene was inactivated by insertion of a chloramphenicol acetyltransferase cartridge and introduced into transformant T14, the resultant corC mutant T14.COR (Fig. 2A) proved to be able to form biofilms as well as its parent strain T14. This finding was pivotal, indicating that the reduction in biofilm formation by mutant T14/27 (Fig. 2A) was not the result of the transposon insertion in the corC gene in T14/27 but was caused by another unidentified factor.

Expression of Hag inhibits biofilm formation. Routine Western blot analysis of the expression of the Hag protein (Fig. 5A) (18, 33) and the UspA1 protein (Fig. 5C) by transformant T14, the corC transposon insertion mutant T14/27, and the corC mutant T14.COR revealed that the corC transposon insertion mutant T14/27 (which did not form a biofilm) expressed the Hag protein whereas the others did not. This finding raised the possibility that the Hag surface protein was somehow interfering with biofilm development in the crystal violet-based assay. Because the hag gene is known to undergo phase variation, likely caused by slipped-strand mispairing involving the poly(G) tract located within the 5' end of its ORF (27, 33; K. Sasaki, K. L. Myers, S. M. Loosmore, and M. H. Klein, Abstr. 99th Gen. Meet. Am. Soc. Microbiol., abstr. B/D-306, 1999), the 5' end of the hag gene from all of these strains was subjected to nucleotide sequence analysis.

Transformant T14 was found to have a hag poly(G) tract that contained 10 G residues (Fig. 5B), resulting in a premature translational stop codon immediately following the poly(G) tract (data not shown). In addition, the hag poly(G) tract from the O46E parent strain was shown to contain 10 G residues (Fig. 5B). In contrast, the 5' end of the hag gene from the corC transposon mutant T14/27 (which expressed Hag) (Fig. 5A) had apparently undergone slipped-strand mispairing and now contained nine G residues (Fig. 5B), which resulted in an intact hag ORF. Nucleotide sequence analysis of the 5' end of the hag gene in transformant T14 revealed that it was identical to that of the hag gene from strain O46E. This result indicated that transformant T14 was likely derived from transformation and allelic exchange involving at least two separate fragments of O46E chromosomal DNA—one containing the 5' end of the uspA1 ORF (described above) and the other containing the 5' end of the hag gene.

To determine directly whether Hag expression adversely affected biofilm formation by transformant T14, we replaced the hag gene of transformant T14 with the wild-type hag gene from strain O35E. To accomplish this, the wild-type hag gene from strain O35E was amplified by PCR and used to transform the spectinomycin-resistant hag mutant T14.HG as described in Materials and Methods. Five transformants that were spectinomycin sensitive were analyzed by Western blotting for Hag expression (Fig. 5A) and were tested for their ability to form biofilms (Fig. 5D). The two transformants (T14.O35EHG1 and T14.O35EHG3) that possessed six G residues in their hag poly(G) tract (Fig. 5B) (resulting in an intact hag ORF) were both positive for Hag expression (Fig. 5A) and markedly impaired in biofilm formation (Fig. 5D).

Biofilm formation by O35E expressing the T14 hybrid UspA1 protein. Because the hybrid O46E-O35E UspA1 protein in transformant T14 was necessary for biofilm formation by that strain, we sought to determine whether expression of this hybrid UspA1 protein would be sufficient to confer biofilm formation ability on strain O35E. Therefore, the hybrid uspA1 gene from transformant T14 was amplified by PCR and used to transform the kanamycin-resistant uspA1 mutant O35E.1 (1). Putative transformants were screened for loss of kanamycin resistance; one of these, designated O35E.14U1 (Fig. 6), was found to contain the entire T14 hybrid uspA1 gene. However, when tested by the crystal violet assay, strain O35E.14U1 was unable to form a biofilm (Fig. 6C). As noted above, the wild-type strain O35E does express the Hag protein (Fig. 6A), which adversely affected biofilm formation by transformant T14. Therefore, the hag gene in O35E.14U1 was inactivated by insertion of a spectinomycin resistance cartridge. The resultant hag mutant O35E.14U1.HG was able to form a biofilm (Fig. 6C). In contrast, a hag mutant of strain O35E (O35E.HG) was shown to be unable to form a biofilm (Fig. 6C). It could be inferred from these results that expression of the T14 hybrid UspA1 protein by strain O35E was necessary but not sufficient to allow biofilm formation in the crystal violet-based assay. The biofilm-forming potential of this hybrid UspA1 protein, however, can only be expressed in this model system in the absence of Hag protein expression.


Figure 6
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  		FIG. 6. Effect of UspA1 and Hag expression on biofilm formation by M. catarrhalis strain O35E and related strains. Expression of Hag (A) and UspA1 (B) was detected by Western blot analysis of whole-cell lysates using MAbs 5D2 and 24B5, respectively, as the primary antibody probes. Molecular mass position markers (in kilodaltons) are shown on the left sides of panels A and B. Biofilm formation ability (C) was assessed by the crystal violet-based assay. Strains used in these experiments include O46E, transformant T14, O35E, hag mutant O35E.HG, O35E.14U1, and hag mutant O35E.14U1.HG.


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DISCUSSION
 
To date, there have been few studies of biofilm formation by the major bacterial pathogens that cause acute otitis media. Some authors have suggested that the persistence of PCR-positive effusions with culture-negative results may be indicative of biofilm formation in the middle ear (6, 35). More direct evidence for biofilm formation by H. influenzae has been obtained from the chinchilla model, where biofilms have been directly visualized in the middle ear cavity by scanning electron microscopy and confocal laser scanning microscopy (8). Murphy and Kirkham (29) were the first to describe biofilm formation by H. influenzae grown in vitro. More recently, Swords and colleagues (39) showed that sialylated lipooligosaccharides promote biofilm formation in vitro by nontypeable H. influenzae, as well as persistence of this organism in the middle ear or lung of animals; Apicella and coworkers reported that biofilms produced by nontypeable H. influenzae contain {alpha}2,6-linked sialic acid (14). Biofilm formation by both S. pneumoniae and M. catarrhalis growing in a cellulose filter support (i.e., the Sorbarod system) has been described, but the identity of the gene products involved in biofilm development was not determined for either organism (3, 33).

Because nothing was known about the M. catarrhalis gene products that might be involved in biofilm formation in the crystal violet-based assay, we sought to use an unbiased approach to this issue. This involved transposon mutagenesis of the biofilm-positive transformant strains T13 and T14, which had been obtained by transformation of the biofilm-negative strain O35E with chromosomal DNA from the biofilm-positive O46E strain. These mutagenesis experiments revealed that the UspA1 protein from these transformants was a hybrid molecule in which the first 155 amino acids were derived from the O46E UspA1 protein, with the remainder being from the O35E UspA1 protein (Fig. 4). These results suggested that the O46E UspA1 protein was involved in biofilm formation by O46E in the crystal violet-based assay; this was confirmed by the reduction in biofilm formation observed with an O46E uspA1 mutant (Fig. 2B). It can also be inferred from these data that the N-terminal quarter of the UspA1 protein from strain O46E is likely involved in the ability of this strain to form a biofilm in the crystal violet-based assay. Interestingly, the UspA1 proteins from both the biofilm-negative strain O35E and the biofilm-positive transformant T14 functioned as adhesins for Chang conjunctival epithelial cells in vitro (Fig. 3). These latter results suggest that UspA1-dependent epithelial cell attachment and biofilm formation by these two M. catarrhalis strains in the crystal violet-based assay are separate and distinguishable activities.

Analysis of the biofilm-negative transposon insertion mutant T14/27 revealed that it possessed a transposon insertion in its corC gene, but an independently constructed corC mutant of transformant T14 was found to be biofilm positive (Fig. 2). This unexpected but pivotal finding had two effects. First, it led us to determine that transformant T14 had likely received a second fragment of O46E DNA containing all or part of the O46E hag gene. This conclusion was based on nucleotide sequence analysis which showed that transformant T14 had a hybrid hag gene, with the 5' portion of the gene coming from O46E and the 3' end of the gene originating from O35E. This hybrid hag gene has a premature translational stop codon in the 5' end of the ORF, which resulted in a lack of expression of the Hag protein by transformant T14. This premature translational stop codon is also present in the O46E parent strain used in this study and is likely the result of slipped-strand mispairing in the poly(G) tract located in the 5' end of the hag gene ORF (27, 33; Sasaki et al., Abstr. 99th Gen. Meet. Am. Soc. Microbiol.). Because we could not be certain that other, undetected transformation events had occurred in transformant T14, the PCR-amplified hybrid T14 uspA1 gene was introduced into strain O35E to create a genetically defined strain that was subsequently used to confirm that expression of the T14 hybrid UspA1 protein was necessary for biofilm formation by strain O35E (Fig. 6). Second, we used this new O35E transformant to construct additional mutants which were used to confirm that expression of the Hag protein prevented or greatly reduced biofilm formation by O35E expressing this hybrid UspA1 protein (Fig. 6).

These studies indicated that the UspA1 protein of strain O46E plays an important role in biofilm formation in the crystal violet-based biofilm assay and that expression of Hag interfered with biofilm development in this model. Both UspA1 (16, 33) and Hag (33) have been shown to be expressed as relatively long, filamentous projections extending from the outer membrane. The UspA1 protein has previously been shown to be an adhesin for Chang conjunctival epithelial cells in vitro (22) and has been reported to bind carcinoembryonic antigen-related cell adhesion molecule 1 (15). Several functions have been assigned to the Hag (i.e., MID) protein, including hemagglutination (33), autoagglutination (33), attachment to type II alveolar epithelial cells (11, 18), and immunoglobulin D-binding activity (12, 33). It should be noted that the crystal violet-based assay likely measures one or more early events in biofilm formation (31, 34). Whether UspA1 or Hag affects the ability of these M. catarrhalis strains to establish or maintain biofilms in continuous flow biofilm systems (3, 39) remains to be determined.


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ACKNOWLEDGMENTS
 
This study was supported by U.S. Public Health Service grant no. AI36344 to E.J.H.

We thank John Nelson, Timothy Murphy, David Goldblatt, Anthony Campagnari, Steven Berk, Frederick Henderson, Richard Wallace, and Merja Helminen for supplying the isolates of M. catarrhalis used in this study.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9048. Phone: (214) 648-5974. Fax: (214) 648-5905. E-mail: eric.hansen{at}utsouthwestern.edu. Back

Editor: J. N. Weiser


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Infection and Immunity, March 2006, p. 1588-1596, Vol. 74, No. 3
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.3.1588-1596.2006
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