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Department of Pediatrics, Center for Microbial Pathogenesis, Columbus Children's Research Institute, The Ohio State University College of Medicine and Public Health, Columbus, Ohio1
Received 17 August 2004/ Returned for modification 29 September 2004/ Accepted 12 November 2004
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionAddendum in proofReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAddendum in proofReferences |
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Twitching motility is a flagellum-independent form of bacterial translocation over moist surfaces and occurs by extension, tethering, and then retraction of polar structures known as type IV pili, or Tfp (6, 29, 43, 51). Tfp are typically 5 to 7 nm in diameter, several micrometers in length, and comprised of a single protein subunit assembled into a helical conformation with five subunits per turn (6, 44). Tfp play a significant role in the pathogenesis of disease caused by Pseudomonas aeruginosa, Vibrio cholerae, Neisseria gonorrhoeae, and numerous other gram-negative pathogens. There are two classes of pilin subunits, type IVa and type IVb, which are distinguished from one another by the average length of both the leader peptide and the mature subunit, by which an N-methylated amino acid occupies the N-terminal position of the mature protein, and by the average length of the D region (for disulfide region) (10). Most of the respiratory pathogens express class IVa pilins, whereas the enteropathogens more typically express class IVb pilins. Regardless of the class of Tfp, expression of these structures has been found to be universally important for both adherence and biofilm formation by many bacteria (24, 27, 29, 35), as well as for virulence of Neisseria species, Moraxella bovis, V. cholerae, enteropathogenic Escherichia coli and P. aeruginosa, among others (26, 27, 35, 42).
Tfp expression is a complex and highly regulated bacterial function. In P. aeruginosa, the biogenesis and function of Tfp are controlled by over 40 genes (42). To date, only a subset of the vast number of related Tfp genes (12, 43) has been found in several members of the HAP (Haemophilus, Actinobacillus, and Pasteurella) family (7, 13, 14, 40), and the expression of Tfp, or a twitching phenotype, has not been described for any H. influenzae isolate. In fact, H. influenzae is classically described as a bacterium that does not express these structures (1, 8, 18, 19), despite the presence of a cryptic gene cluster within the strain Rd genome (16).
Recently, it was recognized that many nontypeable H. influenzae (NTHI) strains form a biofilm (15, 33). Murphy and Kirkham (33) demonstrated that the hif locus, required for the expression of LKP pili (30), was important for biofilm formation by NTHI strain M37. However, strain 86-028NP (the strain used in the present study), like the majority of NTHI isolates (30), does not possess an LKP pilus locus, yet this isolate forms biofilms both in vitro and in vivo (our unpublished observation). Thus, we reasoned that if a pilus-like structure was indeed important to biofilm formation by NTHI, as has been shown in many other bacterial systems (23, 26, 27, 29, 35), there might be another type of pilus responsible for this phenotype. Herein, we identify genes required for Tfp expression, demonstrate that expression of Tfp is dependent on the products of the pilA and comE genes, and demonstrate for the first time twitching motility by H. influenzae.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAddendum in proofReferences |
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Haemophilus strains were grown on chocolate agar or brain heart infusion (BHI) agar supplemented with NAD and heme at a final concentration 2 µg/ml or in a chemically defined medium (9). Media were supplemented with kanamycin at a concentration of 20 µg/ml or spectinomycin at a concentration of 200 µg/ml as appropriate. E. coli strains were grown on Luria-Bertani plates or in Luria-Bertani broth. Where appropriate, media were supplemented with kanamycin at 20 µg/ml and/or with ampicillin at 100 µg/ml.
Recombinant DNA and DNA sequencing methodologies. Restriction enzymes were purchased from New England Biolabs (Boston, Mass.). DNA ligase was purchased from Invitrogen (Carlsbad, Calif.). PCR amplifications were performed with Pfu Turbo (Stratagene, La Jolla, Calif.). Plasmids were purified with QIAGEN (Valencia, Calif.) kits. DNA sequence was determined with an Applied Biosystems 3100 automated sequencer using dye terminator chemistries. Standard methodologies were employed for plasmid constructions. H. influenzae plasmid transformations were performed by electroporation, and mutant constructions were performed by transformation with linear DNA using a modified M-IV method in which cyclic AMP was added to a final concentration of 1 mM after 70 min of incubation in M-IV medium (5), and incubation continued for an additional 30 min to increase transformation efficiency. Sequence comparisons were performed by using the BLAST algorithm (3).
For Southern hybridization, genomic DNA was purified from multiple clinical and laboratory isolates of H. influenzae by using a PUREGENE DNA isolation kit (Gentra Systems, Minneapolis, Minn.). Two micrograms of genomic DNA was digested with MfeI, and the fragments were resolved on a 0.8% agarose gel and then blotted onto a NYTRAN SuPerCharge membrane using a Turbo Blotter kit (Schleicher & Schuell, Keene, N.H.). Membranes were hybridized to a probe generated by PCR amplification of the coding sequence of the 86-028NP pilA gene by using the primers 1 and 2 (Table 1).
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Construction of pilA and comE mutants.
Mutants deficient in expression of either the pilA or comE gene product were constructed in NTHI strain 86-028NP. To accomplish this, the coding sequences of pilA and comE plus an additional 1 kb upstream and downstream were amplified from 86-028NP genomic DNA by PCR using primers 3, 4, 5, and 6, respectively, (Table 1). These products were TA cloned into pGEM-T Easy (Promega Corp., Madison, Wis.) and transformed into E. coli DH10B cells (Invitrogen). Due to the lack of useful restriction sites, it was necessary to introduce an appropriate restriction site into pilA. A BamHI site was introduced into pilA by using a QuikChange Site-Directed Mutagenesis kit (Stratagene Corp.), according to the manufacturer's directions, with primers 7 and 8 (Table 1). The mutagenized plasmid was characterized by sequencing and then digested with BamHI, ligated into a BamHI fragment containing the 
Km-2 element (36), and transformed into One Shot TOP 10 chemically competent E. coli (Invitrogen). A plasmid with the correct restriction map was identified, linearized with MluI, and then transformed into strain 86-028NP by using a modified M-IV transformation protocol (5, 28). Transformants were selected on chocolate agar supplemented with kanamycin, and allelic exchange was verified by Southern analysis.
Similarly, a BamHI site was constructed in the comE gene by using primers 9 and 10 (Table 1), employing the methodology described for the pilA construction. A comE mutant in strain 86-028NP was then constructed by using the strategy described above for the construction of the pilA mutant.
Complementation was performed by using a derivative of the shuttle vector pGZRS-39A (46), in which the kanamycin resistance gene was replaced by the spectinomycin resistance gene from pSPECR (48). The pGZRS-39A plasmid backbone, without the kanamycin resistance gene, was amplified by using primers 11 and 12 (Table 1). Both primers had BglII sites at their 5' ends. The amplicon was digested with BglII and ligated into the spectinomycin gene that had been isolated from pSPECR after BglII digestion. After transformation into H. influenzae strain Rd, spectinomycin-resistant clones were selected, plasmids were characterized, and a plasmid with the correct restriction map was saved as pSPEC1. We then amplified the pilABCD gene cluster using primers 13 and 14 and the comEF genes using primers 15 and 16 (Table 1). The amplicons were cloned into pCR Blunt II TOPO (Invitrogen), and plasmids with the correct inserts were saved. The pilABCD fragment was released as an EcoRI fragment and cloned into pSPEC1 that had been digested with EcoRI. The comEF fragment was cloned as a BamHI-EcoRI fragment into pSPEC1 that had been digested with the same enzymes. After transformation of the ligation mixtures into H. influenzae strain Rd, plasmids with the correct restriction maps were saved as pPIL1 and pCOM1 and transformed into strain 86-028NP and subsequently into strains 86-028NP pilA::kan and 86-028NP comE::kan, thus generating the complemented strains 86-028NP pilA::kan(pPIL1) and comE::kan(pCOM1), respectively.
Negative staining and transmission electron microscopy (TEM). NTHI strain 86-028NP and its pilA or comE mutant were inoculated onto either chocolate agar, supplemented BHI agar, or chemically defined agar plates (at pH 7.2 or 8.5 to 9.0) and incubated for 24 h at 37°C and 5% CO2. Bacteria were scraped from agar plates, suspended in 10 µl of sterile water, and then negatively stained with an equal volume of a Whatman-filtered solution containing 2.0% (wt/vol) ammonium acetate (Sigma, St. Louis, Mo.) and 2.0% (wt/vol) ammonium molybdate (Sigma) in sterile water (4). Formvar- and carbon-coated copper grids (300 mesh; Electron Microscopy Sciences, Hatfield, Pa.) were placed under the droplet. After 5 min, grids were blotted and allowed to air dry overnight prior to viewing with a Hitachi model H-600 transmission electron microscope with an attached video monitor (Gatan, Inc., Pleasanton, Calif.) and digital imaging system (Gatan, Inc.).
For blinded TEM evaluation of the 86-028NP parental isolate and its pilA mutant, negative stains were prepared as described above, and multiple unlabeled grids were provided (to L.O.B.). For each grid, a minimum of 15 individual grid squares were evaluated at 
x25,000 magnification. Within each of these 15 grid squares, a minimum of eight bacterial cells were scanned around their entire periphery for the presence of Tfp-like structures. Thus, a minimum of 120 individual NTHI isolates were evaluated per grid. Grids were also evaluated for the presence of bundles of free pili.
Motility assay. NTHI strain 86-028NP and its pilA or comE mutant were inoculated onto chemically defined 1% agar slabs (3 mm in depth), pH 9.0, and placed onto sterile microscope slides. A 0.2-µl droplet containing 109 CFU of NTHI/ml of saline was dropped onto the agar slab, covered with a sterile glass coverslip, and incubated at 37°C, 5% CO2, in a humidified atmosphere (11). The outermost edge of the inoculum was examined hourly for up to 24 h with a Zeiss Axioskop 40 microscope under a x40 objective. Images were recorded with a Zeiss Axiocam MRC digital camera, and the assay was repeated a minimum of four times on different days.
The complemented strains 86-028NP pilA::kan(pPIL1) and 86-028NP comE::kan(pCOM1) were evaluated in an identical manner.
Nucleotide sequence accession numbers. The pil and com gene cluster sequences from strain 86-028NP have been submitted to GenBank with accession numbers AY816324 and AY816325, respectively, and the pilA gene sequences have been submitted to GenBank with accession numbers AY818310 to AY818319.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAddendum in proofReferences |
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In other Tfp systems, proteins designated as secretins have been characterized as those that form gated channels in the outer membrane through which the pilus is assembled. The Tfp secretin in P. aeruginosa is the PilQ protein (29). We employed the BLAST algorithm to identify the homologue of PilQ in the 86-028NP genome. PilQ is 31% identical to a putative 445-amino-acid protein produced by strain 86-028NP. In strain 86-028NP, this protein is the homologue of the ComE protein of H. influenzae strain Rd (HI0435), a protein known to be required for transformation. In both strains 86-028NP and Rd, the com gene cluster contains six genes. We have retained the strain Rd nomenclature, thus designating strain 86-028NP genes as comABCDEF. A map of the pil and com gene clusters from strain 86-028NP is shown in Fig. 1.
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The pilABCD gene cluster was amplified and cloned into the shuttle vector pSPEC1 to form the plasmid pPIL1, and strain 86-028NP pilA::kan(pPIL1) was constructed as described in Materials and Methods. When grown in chemically defined medium at alkaline pH, this strain produces Tfp, verifying that the mutation in the pilA gene in strain 86-028NP pilA::kan is responsible for the pilus-negative phenotype (Fig. 5A and B).
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The comEF genes were amplified and cloned into pSPEC1 to form the plasmid pCOM1, and strain 86-028NP pilA::kan(pCOM1) was constructed as described in Materials and Methods. When grown in chemically defined medium at alkaline pH, this strain produced Tfp (Fig. 5C and D), which verifies that the mutation in the comE gene in strain 86-028NP comE::kan was responsible for the altered pilus phenotype described above.
Characterization of twitching phenotype. In addition to demonstrating that NTHI expresses Tfp under defined growth conditions, we have also generated data to demonstrate that the Tfp expressed by NTHI are functional. After 7 h, the outermost edge of the zone of growth surrounding a point of inoculation of the parental isolate demonstrated a rugous and ruffled appearance (Fig. 6A) that was highly characteristic of the motile rafts of twitching described for P. aeruginosa (11), whereas the outermost area of bacterial growth surrounding the point of inoculation of the pilA mutant was smooth (Fig. 6B). The comE mutant demonstrated an intermediate phenotype characterized by an edge with a slightly ruffled appearance (Fig. 6C). These growth phenotypes remained constant after 24 h, with the parental isolate demonstrating a rugous appearance with motile rafts of cells at the leading edge (Fig. 6F). The pilA mutant remained relatively smooth along the outermost edge from the point of inoculation (Fig. 6G), and the comE mutant maintained an intermediate phenotype (Fig. 6H). These observations were readily reproducible and were thus a highly characteristic growth phenotype for these bacteria.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAddendum in proofReferences |
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As mentioned in Results, we investigated this hypothesis further and found a single pilA gene in the genome of all clinical H. influenzae strains analyzed. Interestingly, the NTHI PilA proteins appear to represent a new class of Tfp. At 12 residues, the leader peptide is larger than that characteristic for type IVa pilins (typically 5 to 6 residues in length) and yet shorter than the typical IVb leader peptide (15 to 30 residues). Similarly, at 137 residues, the mature NTHI pilin is shorter than either class IVa or IVb pilins (150 and 190 residues, respectively). Since the NTHI PilA proteins begin with an N-methylated phenylalanine, they are more like class IVa pilins; however, in electron micrographs, free NTHI Tfp always appear in laterally associated bundles (Fig. 4B), a phenotype more classically associated with class IVb pilins due to their ability to self-associate through antiparallel interactions (10).
In terms of NTHI PilA sequence diversity, despite areas wherein one or two amino acids are variant, these sequences are highly homologous overall. Two areas of potentially important diversity exist at positions 55 to 64 and 79 to 87. Within the first region, among the clinical isolates, there appears to be two major variants, one representing the majority (7 of 11 isolates [64%]) and characterized by the sequence NET/ITNCT/MGGK, and the other representing the minority (4 of 11 isolates [36%]) and characterized by the sequence GKP/LST/SCSGGS. There are, however, some additional minor variations at positions +57 and +61 in the majority grouping and at positions +57 and +59 in the minority grouping. The diversity noted at position +61 has been seen only in one isolate to date (strain 1885MEE), wherein there is a T-to-M substitution. Within the second focused region of diversity (positions 79 to 87), there appears to be two equally distributed variants among the clinical NTHI isolates. The sequence ASVKTQSGG is present in 5 of 11 clinical isolates (
45%), whereas the sequence KSVTTSNGA is present in 6 of 11 clinical isolates (55%).
In addition to characterizing the product of the pilA gene, we also interrogated our contig set (generated during our efforts to sequence the genome of strain 86-028NP [see http://www.microbial-pathogenesis.org]) with the sequences for both the Tfp retraction protein, PilT, and the secretor protein, PilQ, as well as other Tfp proteins of P. aeruginosa in order to identify homologues of the genes encoding these proteins in the 86-028NP genome. We found that PilB expressed by strain 86-028NP is a homologue of PilB, PilT, and PilU. PilT is required for twitching motility and is the pilus retraction protein of both N. meningitidis (1, 19) and N. gonorrhoeae (50, 51). The product of the comE gene, a protein required for transformation in H. influenzae, was found to have homology to PilQ of P. aeruginosa (29). PilQ is a member of the pIV/PulD family, which in multimeric form acts as a gated channel through which macromolecules such as pilin subunits pass (29, 50, 51). Recognition that ComE has homology to secretin proteins suggests that the products of the com gene cluster may be involved in the biogenesis of the type IV pilus we have described here.
Collectively, these data suggested to us the tremendous likelihood that NTHI could not only express functional Tfp, it could also potentially twitch. This was an extremely exciting hypothesis, as historically, while it is well known both that H. influenzae is naturally competent and that Tfp are classically associated with competence, type IV pili had heretofore never been demonstrated in H. influenzae. Transmission electron microscopy provided direct evidence that strain 86-028NP did indeed express intact type IV pilus-like structures on its surface when grown in a defined RPMI 1640-based medium at alkaline pH. The relative size and distribution of type IV pili by individual bacterial cells were highly analogous to those described for the closely related family member P. multocida (38). These structures were not observed when NTHI was grown in rich supplemented BHI medium or in chemically defined medium at neutral pH. A mutant deficient in PilA did not produce these surface structures, whereas a ComE mutant produced large appendages that appeared to be membrane bound and to contain a fibrous material, terminating in a bulbous tip and often demonstrating a regular pattern of constriction and expansion. These structures were highly analogous to those produced by an N. meningitidis pilQ/pilT mutant as shown by Wolfgang and colleagues (51).
We also present several pieces of data that support the assertion that NTHI twitches. When grown on defined medium at an alkaline pH, NTHI strain 86-028NP demonstrated classic rafts of cells at the leading edge of bacterial growth from a point of inoculation, similar to that reported for P. aeruginosa (39), when grown under conditions of nutrient depletion. The pilA and comE mutants did not form these motile rafts, whereas the complemented mutants were shown to regain the parental phenotype.
In summary, we have identified and characterized genes that encode expression of functional type IV pili, and we have presented both direct and indirect data in support of the expression of functional Tfp by H. influenzae. The fact that Tfp were expressed under alkaline conditions is particularly intriguing since the pHs of both serous and mucoid effusions recovered from patients with chronic OM are uniformly alkaline (47). Alkalinity within middle ear effusions may thus represent an environmental cue that contributes to the regulation of expression of virulence factors, such as Tfp, by NTHI. In addition to providing a form of motility, type IV pili are involved in transformation competence in many bacteria (1, 18, 19, 22, 45) including H. influenzae and may be important for biofilm formation by NTHI as well. Importantly, Tfp have been shown to play a role in pathogenesis, as they are essential for adherence to human epithelial cells by N. gonorrhoeae (49) and P. aeruginosa (2) as well as for meningeal invasion by N. meningitidis (34). We know that NTHI forms biofilms in the middle ear during experimental OM (15, 37), a phenotype that is often associated with twitching motility (24, 26, 29, 35). Therefore, studies designed to define the role of Tfp in competence and the pathogenesis of NTHI-induced OM and/or biofilm formation in the middle ear are under way.
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ADDENDUM IN PROOF |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAddendum in proofReferences |
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8e9 CFU of nontypeable Haemophilus influenzae) was placed in the center of a slab of chemically defined agar (pH 9.0) and overlaid with a sterile glass coverslip. After incubation for 24 h at 37°C in a humidified atmosphere of 5% CO2, video images were captured at 4 frames/s with a 63x objective with a 2:1 digital zoom. The video clip is available at http://www.microbial-pathogenesis.org/H.influenzae.86028/.
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ACKNOWLEDGMENTS |
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We thank Brian Quist and Cindy McAllister for excellent technical assistance and Jennifer Neelans for manuscript preparation. We also thank Susan West (University of Wisconsin) and Daniel Morton (University of Oklahoma Health Sciences Center) for the plasmids pG2RS-39A and pSPECR, respectively.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Pediatrics, Columbus Children's Research Institute, Center for Microbial Pathogenesis, The Ohio State University College of Medicine and Public Health, 700 Children's Dr., Columbus, OH 43205-2696. Phone: (614) 722-2915. Fax: (614) 722-2818. E-mail for Lauren O. Bakaletz: BakaletL{at}pediatrics.ohio-state.edu. E-mail for Robert S. Munson, Jr.: MunsonR{at}pediatrics.ohio-state.edu.
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