INTRODUCTION

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Haemophilus influenzae strains are gram-negative rods which colonize human respiratory and genital mucosa. Although they are commensal bacteria, they can cause serious respiratory tract infections, meningitis, and genital and neonatal infections. Adhesion to epithelial cells is the first step in host colonization by many bacteria (5, 9). Adhesion can be mediated by nonfimbrial and fimbrial structures. Most nontypeable strains of Haemophilus (95%) adhere to human epithelial cells either by high-molecular-weight surface proteins (84%) or by Hia protein (16%) (4, 34, 35). Short, thin surface fibrils involved in H. influenzae type b (Hib) adherence to human epithelial cells were recently identified (31, 32). Both Hib and nontypeable H. influenzae (NTHi) strains commonly express fimbriae, which are polymeric structures composed of a major structural protein associated with several other minor proteins. The fimbrial proteins of a large number of gram-negative bacteria have been purified, and their sequences have been determined. The major fimbrial proteins from different species are often very similar (14, 15, 36, 38). Fimbria-mediated adhesion to cells has also been reported for H. influenzae strains that colonize the nasopharynx and that are responsible for various infections, including meningitis, chronic bronchitis, otitis media, and Brazilian purpuric fever (3, 12, 21, 29, 37).

The genes coding for the major fimbrial subunit (hifA genes) in several Hib strains and in some NTHi strains responsible for various diseases have been characterized. The deduced amino acid sequences of these proteins are about 80% similar, and the amino acid sequences are particularly well conserved at the N- and C-terminal ends (2, 7, 10, 15, 33, 38, 42). The genes from some Hib strains coding for minor proteins that constitute fimbriae (hifD and hifE genes) have been characterized (18, 39). These two genes map downstream from the hifA gene. The complete fimbrial gene cluster in Hib has been identified and lies between the purE and pepN genes (11, 39). The adhesive component in H. influenzae fimbriae has not been clearly defined; it could be the major subunit (HifA) or HifE, a minor subunit located at the tips of fimbriae (17, 40).

Another fimbrial gene in a strain of NTHi has been characterized. It encodes a fimbrin protein, an adhesin homologous to OmpA proteins of many gram-negative bacteria (30).

In the last 20 years, urogenital, mother-infant, and neonatal infections caused by Haemophilus strains have been reported with increasing frequency (1, 16, 23, 41). The members of a group of Haemophilus strains isolated from neonatal and genital tract infections (referred to here as genital strains) and commonly identified as H. influenzae biotype IV have several unusual features (19, 24, 26). Genetic analysis of these strains demonstrates that they constitute a cryptic genospecies which forms a monophyletic unit with H. influenzae and Haemophilus haemolyticus, most closely related to H. haemolyticus (25, 27). These isolates may have a specific tropism for the genital tract. They were not detected among a large number of H. influenzae isolates recovered from invasive, respiratory, and other infections in children and adults (1, 20). Most express peritrichous fimbriae, adhere better to HeLa cells than to HEp-2 cells and do not cause agglutination of human erythrocytes expressing the AnWj antigen (28), contrasting with numerous fimbriated H. influenzae strains responsible for respiratory infections and meningitis (3, 12, 21, 37). Therefore, if these fimbriae are involved in the colonization of the genital tract, they may differ from those produced by strains isolated from the respiratory tract.

Our objective was to explore a group of 19 strains genetically assigned to the cryptic genital Haemophilus genospecies (27) for the presence of fimbrial genes. In addition, this population was investigated for the presence of the gene coding for fimbrin. When present, these genes were sequenced, and the sequences were compared to those of previously described genes coding for proteins that are components of the fimbriae of H. influenzae. If no such gene was detected, the absence of these genes was confirmed by Southern blotting experiments and the nucleotide sequence between the purE and pepN genes was analyzed. For each isolate, the expression and the morphology of fimbriae were studied by electron microscopy.

	MATERIALS AND METHODS

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Bacterial strains. Thirty-one Haemophilus strains were studied, including 1 H. influenzae biotype I strain, 1 H. influenzae biotype II strain, 1 H. influenzae biotype III strain, 6 H. influenzae biotype IV sensu stricto strains, 2 H. haemolyticus strains, 1 H. influenzae biogroup Aegyptius strain, and 19 strains previously assigned to the cryptic genital Haemophilus genospecies on the basis of DNA-DNA hybridization and small-subunit ribosomal DNA sequencing (25, 27). Genital strains of Haemophilus from the United States were kindly provided by J. M. Musser (Department of Pathology, Baylor College of Medicine, Houston, Tex.). The anatomic and geographic origins of these strains are reported in Table 1. Strains were stored at 80°C in Schaedler-vitamin K₃ broth (bioMérieux, Marcy l'Etoile, France) with 10% glycerol. A heavily fimbriated acapsular variant of H. influenzae (strain 770235), kindly provided by L. van Alphen (Department of Medical Microbiology, University of Amsterdam, Amsterdam, The Netherlands), was used as a control. This strain harbors long, thick, hemagglutination-positive (LKP) pili. The nucleotide sequences of the genes coding for proteins constituting the fimbriae of this strain have been reported by van Ham et al. (39).

Electron microscopy. The presence and appearance of fimbriae were examined by electron microscopy before and after selection of piliated Haemophilus strains as previously described (28). Bacteria grown to stationary phase were washed three times in saline buffer, settled onto 400-mesh copper grids coated with carbon film, negatively stained with a 1.5% uranyl acetate solution in distilled water as previously described (28), and then examined with a JEOL 1010 electron microscope at 80 kV.

Genomic DNA extraction. Each strain was subcultured on four chocolate agar plates (15 by 15 cm) for 18 to 24 h at 37°C in 8% CO₂. The cultures were checked visually for purity, harvested in 15 ml of buffer (40 mM Tris, 2 mM EDTA, pH 8), and lysed by adding 150 µl of a 25% (wt/vol) aqueous solution of sodium dodecyl sulfate and 22.5 µl of 2% self-digested pronase (Sigma, St. Louis, Mo.). The mixture was incubated overnight at 37°C, and DNA was extracted and purified as previously described (6), dialyzed on 0.025-µm-pore-size filters (Millipore, St Quentin en Yvelines, France), and diluted in water to a concentration of 10 µg/ml.

PCR assay. One hundred nanograms of DNA was used for PCR. Eleven primers were used to amplify genes coding for a major fimbrial subunit (Eurogentec, Seraing, Belgium) (Tables 2 and 3). Primers N1, N34, N51, N69, C189, C198, and C207 were designed to correspond to conserved sequences in the hifA genes of Hib 770235, AO2, and Eagan, NTHi M37 and 1128, and H. influenzae biogroup Aegyptius, described previously (Fig. 1 and Table 2) (2, 8, 10, 39, 42). Primers were named as follows: the letters N and C indicate the localization of the primer sequence in the 5' and 3' ends of the gene, respectively, and the number following the letter corresponds to the position of the first or the last translated codon, respectively (Fig. 1). The Crypt N and Crypt C primers were designed to correspond to two regions that are different in the major fimbrial protein genes of genital Haemophilus strains and of H. influenzae (Fig. 1 and Table 2). Primers fim5 and fim3 were deduced from a gene coding for the fimbrial subunit (fimbrin) of the NTHi 1128 strain sequenced by Sirakova et al. (30) (Table 3).

View larger version (26K): [in this window] [in a new window]   FIG. 1.   Amino acid sequence deduced from major fimbrial protein gene sequence of genital Haemophilus strain. The sequence is compared with previously reported HifA sequences (2, 8, 13, 38, 42). Amino acid residues chosen as the basis of primer sequences are double underlined, and the name of each primer is shown above the line. Dots represent identity. Dashes indicate gaps introduced to optimize alignment. H. Aegyptius; H. influenzae biogroup Aegyptius; genital H., genital cryptic strains of Haemophilus.

DNA sequencing. PCR products were purified by a 15-min spin at 500 × g on a Microcon 100 (Amicon, Beverly, Mass.) to remove unincorporated deoxynucleoside triphosphates, primers, and salts. Nucleotide sequences were determined by the dideoxy chain termination method of Sanger et al. (29a) with Thermo Sequenase dye terminator cycle sequencing premixed version 2 (Amersham, Les Ulis, France) and PCR primers with an Abi Prism 377 sequencer (Perkin-Elmer) according to the manufacturer's instructions.

Southern blotting. To confirm the absence of major or minor fimbrial protein genes for strains which did not give a PCR product with the fimbrial primers, probes for major and minor fimbrial protein genes labeled with digoxigenin (DIG) were synthesized by PCR with alkali-labile DIG-11-2'-deoxyuridine-5'-triphosphate (DIG-11-dUTP) (Boehringer). For this PCR assay 30 µM dTTP was replaced by 30 µM DIG-11-dUTP. One probe was prepared with the N1 and C207 primers and DNA from strain 11PS, which carries the hifA-like gene observed in genital strains. Another probe was constructed with the N1 and C207 primers and the Hib reference strain 770235. Two other probes were prepared with a genital strain of Haemophilus: the first was prepared from strain 11PS by using the hifD5 and hifD3 primers, and the second was prepared from strain 26E by using the hifE5 and hifE3 primers.

Nucleotide sequence accession numbers. The sequence of the genital Haemophilus major fimbrial protein gene (ghfA) has been deposited in the EMBL sequence database under accession no. AJ000653. The accession numbers of the hifA-like gene sequences of Hib strain 5494 and H. influenzae biogroup Aegyptius strain 52129 are AJ000636 and AJ000637, respectively. The accession numbers corresponding to the genes coding for minor fimbrial proteins are AJ006783 for the ghfD gene of the genital strains 15N, 10U, 11PS, 26E, PIZ, and 2406 and AJ006784 for the ghfE gene of the genital strain 26E. The accession number of the omp gene of the genital strain 16N is AJ007317.

	RESULTS

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Characterization of a genital Haemophilus major fimbrial gene with primers corresponding to conserved sequences in hifA genes of Hib. PCR assay with the primers N34 and C207 (Table 2; Fig. 1) amplified a fragment of about 550 bp from DNA of the control strain (Hib 770235). A comparable fragment was obtained from 6 of the 19 genital Haemophilus strains. These six strains were all from French patients; five were from the genital tracts of adult patients (strains 10U, 11PS, 26E, PIZ, and 2406), and only one was from a neonate (strain 15N) (Table 1). A fragment was also amplified from two H. influenzae reference strains: the Hib CIP 5494 strain, which is of biotype IV sensu stricto, and the H. influenzae biogroup Aegyptius CIP 52129 strain (Table 1). Except for CIP 52129, all strains for which a fragment was amplified exhibited peritrichous piliation under our culture conditions (Table 1).

View larger version (39K): [in this window] [in a new window]   FIG. 2.   PCR products obtained with fimbrial primers corresponding to the extreme ends of the three genes coding for the three proteins constituting the fimbria. (A) Primers N1 and C207 were used to amplify a hifA-like gene fragment. The fragment amplified from the control strain Hib 770235 (lane 7) had a slightly lower electrophoretic mobility than the fragments obtained from genital Haemophilus strains (lanes 1 to 6). (B) PCR products obtained with hifD5 and hifD3. (C) PCR products obtained with hifE5 and hifE3. Lanes 1 to 6, genital cryptic Haemophilus strains 15N, 10U, 11PS, 26E, PIZ, and 2406, respectively; lane 7, Hib control strain 770235; lane L, 100-bp ladder.

View larger version (124K): [in this window] [in a new window]   FIG. 3.   Southern blots obtained with the major fimbrial protein gene of genital Haemophilus (ghfA) labeled with DIG as a probe. Hybridization was observed only with DNA from strains giving a PCR product with primers N1 and C207 (Hib strains 770235 and 5494, H. influenzae biogroup Aegyptius strain 52129, and genital Haemophilus strains 15N, 10U, 11PS, 26E, PIZ, and 2406). Lanes 1 to 7, genital cryptic Haemophilus strains 15N, 16N, 10U, 11PS, 26E, PIZ, and 2406, respectively; lane 8, Hib control strain 770235; lanes 9 and 10, Hib strain CIP 5494 and H. influenzae biogroup Aegyptius strain CIP 52129, respectively.

Identification of genes coding for minor fimbrial proteins for genital Haemophilus. Two sets of primers were used to amplify the genes coding for the two minor fimbrial proteins. The hifD5 and hifD3 primers (Table 3), corresponding to the extreme ends of the hifD gene of the Hib strain 770235, were used to amplify the hifD-like gene, and hifE5 and hifE3 (Table 3), corresponding to the extreme ends of the hifE gene of the control strain, were used to amplify the hifE-like gene.

Analysis of the region between the purE and pepN genes for ghfA-negative strains. By using primers derived from the conserved flanking regions (purE and pepN genes), a PCR fragment of about 450 bp was obtained for all ghfA-negative genital strains of Haemophilus, except for two (genital strains 1595 and 3N) for which the PCR fragment was of about 250 bp. A 250-bp fragment was also amplified for the two strains of H. haemolyticus (CIP 102348 and CIP 103290).

Characterization of a gene in our genital Haemophilus collection possibly involved in adherence and previously described as a fimbrial gene in an NTHi strain. The primers fim5 and fim3 (Table 3) were deduced from the sequence of the gene coding for the fimbrin previously studied by Sirakova et al. (30). A fragment of about 1.1 kb was amplified for all genital strains of Haemophilus, for the 10 strains of H. influenzae sensu stricto, for the strain of H. influenzae biogroup Aegyptius, and for a strain of H. haemolyticus. Only one strain of H. haemolyticus (strain 102348) was negative. This PCR fragment was sequenced for the genital strain 16N. The sequence of the fimbrial gene in an NTHi strain (strain 1128) described by Sirakova et al. (30) is 84% identical to the sequence of the PCR fragment from 16N. A search for homology by using the FASTA program indicated that the fimbrin-like proteins encoded by these genes were similar to several H. influenzae outer membrane P5 proteins.

Electron microscopic observation of the fimbriae. Fourteen of the 19 genital Haemophilus strains showed abundant peritrichous piliation (Table 1). There was no notable difference between the appearances of the fimbriae on strains having LKP-like fimbrial genes and those on the genital strains lacking these genes. The appearances of fimbriae before or after several contacts with human erythrocytes were the same. Nevertheless, two of the five nonpiliated strains (189 and 3N) exhibited fimbriae after this enrichment. No evident difference was observed between the electron microscopic appearances of these fimbriae and those of the 14 other strains.

	DISCUSSION

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The aim of this study was to identify and sequence genes coding for proteins participating in fimbria biogenesis in a particular group of Haemophilus strains that are specifically associated with genital, maternofetal, and neonatal infections.

As previous studies demonstrate about 80% similarity between the primary sequences of the major fimbrial subunits in all Hib and NTHi strains, including H. influenzae biogroup Aegyptius (2, 7, 10, 42), we used primers corresponding to conserved sequences at the 3' and 5' ends of previously described hifA genes. A hifA-like gene (named ghfA) was found in only 6 of the 19 genital Haemophilus strains studied. These PCR results were confirmed by Southern blotting with the hifA and ghfA genes as probes. This gene had 71 to 80% identity with the known hifA genes of various Haemophilus strains, percentages of identity which are consistent with those observed between H. influenzae genes encoding the major fimbrial subunit (2, 7, 10, 42). No amplification was obtained with the two H. haemolyticus strains, although this genospecies is the most closely related to genital Haemophilus genospecies strains (25, 27). ghfA codes for a protein (GhfA) whose sequence has 63% identity and 71% similarity to the translated amino acid sequence of the Hib 770235 hifA gene (38). It is 3 to 9 amino acids shorter than known HifA proteins in Haemophilus strains and includes two cysteines, 40 residues apart, and a penultimate tyrosine, a characteristic of H. influenzae HifA proteins (2, 8, 10, 13, 38, 42). Differences between the amino acid sequences were not localized to any one region but were distributed throughout the protein. In addition, the FASTA software (GenBank) found an identity of 62 to 68% between GhfA and major fimbrial subunit precursors of several H. influenzae strains. Therefore, GhfA appears to be a member of a family of major fimbrial proteins which includes almost all HifA proteins characterized to date from Hib, NTHi, and H. influenzae biogroup Aegyptius strains. There are also identities of 35% with the Escherichia coli F17 fimbrial protein precursor and 36% with the Klebsiella pneumoniae fimbrial subunit type 3 precursor.

Geluk et al. suggest that the complete fimbria gene cluster is either always present or entirely absent (11). Southern blotting suggest the presence of ghfE (a hifE-like gene) in the six genital Haemophilus strains which possess ghfA and ghfD. Nevertheless, at least one of the extreme ends (hifE5 or hifE3 sequences) of the gene in five of these six strains differs, because no amplification was obtained with these primers.

For 13 of the 19 genital strains, no PCR product was obtained with any of the primers used. This result was confirmed by Southern blotting with the three fimbrial gene probes and by sequencing of the products amplified with primers defined on the two genes surrounding the fimbrial cluster (purE and pepN). These data confirm the absence of LKP-like fimbrial genes from most (68%) of the genital Haemophilus strains. However, 10 of these 13 genital Haemophilus strains were piliated (Table 1). Therefore, other genes coding for proteins that constitute these fimbriae must be located elsewhere on the chromosome.

Because genital Haemophilus strains are commonly phenotypically identified as H. influenzae biotype IV, we also sequenced the PCR fragment of an H. influenzae biotype IV sensu stricto strain (Hib CIP 5494) for comparison. The nucleotide sequence is 69% identical to that of ghfA. The translated sequences are 63% identical and 65% similar. These percentages correspond to the identities and similarities observed between GhfA and H. influenzae HifA proteins as assessed by using FASTA software.

van Ham et al. demonstrated that HifA mediated H. influenzae-specific adherence (40). They found that the hydrophilicity pattern of HifA was different from that of HifD, which is a minor subunit of fimbriae and is closely related to HifA but which is not required for adherence. They suggested, therefore, that hydrophilic domains of HifA are responsible for binding to a specific eukaryotic receptor. Recently, McCrea et al. (17) suggested that the adhesion is mediated by the HifE protein localized at the tips of fimbriae rather than by the HifA major subunit, because hemagglutination by their Hib Eagan strain was inhibited by the presence of antibodies directed against HifE. Cryptic genital Haemophilus strains do not agglutinate erythrocytes (28). Nevertheless, the three hydrophilic domains previously observed in HifA were also identified in GhfA from the cryptic Haemophilus strains. This suggests that the three hydrophilic domains of HifA may be involved in an interaction other than binding to a specific eukaryotic receptor and that the adhesive site may not be localized on the HifA protein. These observations are in part in agreement with a previous study (2) in which an LKP hifA-like gene was amplified by PCR from a strain responsible for otitis that did not agglutinate erythrocytes. In this strain, pili were morphologically different from LKP pili, and an additional fimbrial subunit gene (fimbrin gene) having a low sequence similarity with the LKP gene has since been identified by Sirakova et al. (30). By using primers derived from this fimbrin gene, an omp gene which has 84% identity with the fimbrin gene described by Sirakova et al. and about 85% identity with the ompP5 genes of many H. influenzae strains was amplified for all our genital strains. Thus, the product of this gene could be an adhesin commonly observed for Hib, NTHi, and genital strains of Haemophilus, but it seems doubtful that it is the major protein constituting fimbriae. Therefore, other genes, as yet unidentified, may explain the piliation observed by electron microscopy on genital Haemophilus strains which do not carry LKP-like fimbrial genes.

Genital Haemophilus strains that possess the ghfA and ghfD genes were isolated more often from mucosal cells of adults (5 of the 6 strains) than from neonates (only 1 of the 13 strains). In addition, ghfA and ghfD were present only in French strains. Previous genetic studies give no evidence for differences between French and U.S. strains (25, 27). Therefore, the apparent relationship between the geographic origin of strains and the presence of ghf genes may reflect the fact that most strains isolated from urogenital epithelial cells were from French adults, whereas most of the strains isolated in the United States were from neonates. Thus, these fimbrial genes may confer on the strain the ability to adhere to urogenital epithelial cells and to persist in the ecological and physiological conditions of the adult genital tract. In contrast, ghfA and ghfD do not appear to be essential for colonizing neonates at birth. The study of a larger number of strains may confirm this observation.