Cloning and characterization of the lipooligosaccharide galactosyltransferase II gene of Haemophilus ducreyi

Haemophilus ducreyi is the etiologic agent of chancroid, a genital ulcer disease. The lipooligosaccharide (LOS) is considered to be a major virulence determinant and has been implicated in the adherence of H. ducreyi to keratinocytes. Strain A77, an isolate from the Paris collection, is serum sensitive, poorly adherent to fibroblasts, and deficient in microcolony formation. Structural analysis indicates that the LOS of strain A77 lacks the galactose residue found in the N-acetyllactosamine portion of the strain 35000HP LOS as well as the sialic acid substitution. From an H. ducreyi 35000HP genomic DNA library, a clone complementing the defect in A77 was identified by immunologic screening with monoclonal antibody (MAb) 3F11, a MAb which recognizes the N-acetyllactosamine portion of strain 35000HP LOS. The clone contained a 4-kb insert that was sequenced. One open reading frame which encodes a protein with a molecular weight of 33,400 was identified. This protein has homology to glycosyltransferases of Haemophilus influenzae, Haemophilus somnus, Neisseria species, and Pasteurella haemolytica. The putative H. ducreyi glycosyltransferase gene was insertionally inactivated, and an isogenic mutant of strain 35000HP was constructed. The most complex LOS glycoform produced by the mutant has a mobility on sodium dodecyl sulfate-polyacrylamide gel identical to that of the LOS of strain A77 and lacks the 3F11-binding epitope. Structural studies confirm that the most complex glycoform of the LOS isolated from the mutant lacks the galactose residue found in the N-acetyllactosamine portion of the strain 35000HP LOS. Although previously published data suggested that the serum-sensitive phenotype of A77 was due to the LOS mutation, we observed that the complemented A77 strain retained its serum-sensitive phenotype and that the galactosyltransferase mutant retained its serum-resistant phenotype. Thus, the serum sensitivity of strain A77 cannot be attributed to the galactosyltransferase mutation in strain A77.     

Bacteriophage HP2 of Haemophilus influenzae

Temperate bacteriophages effect chromosomal evolution of their bacterial hosts, mediating rearrangements and the acquisition of novel genes from other taxa. Although the Haemophilus influenzae genome shows evidence of past phage-mediated lateral transfer, the phages presumed responsible have not been identified. To date, six different H. influenzae phages are known; of these, only the HP1/S2 group, which lyosogenizes exclusively Rd strains (which were originally encapsulated serotype d), is well characterized. Phages in this group are genetically very similar, with a highly conserved set of genes. Because the majority of H. influenzae strains are nonencapsulated (nontypeable), it is important to characterize phages infecting this larger, genetically more diverse group of respiratory pathogens. We have identified and sequenced HP2, a bacteriophage of nontypeable H. influenzae. Although related to the fully sequenced HP1 (and even more so to the partially sequenced S2) and similar in genetic organization, HP2 has a few novel genes and differs in host range; HP2 will not infect or lysogenize Rd strains. Genomic comparisons between HP1/S2 and HP2 suggest recent divergence, with new genes completely replacing old ones at certain loci. Sequence comparisons suggest that H. influenzae phages evolve by recombinational exchange of genes with each other, with cryptic prophages, and with the host chromosome.     

Proteome of Haemophilus ducreyi by 2-D SDS-PAGE and mass spectrometry: strain variation, virulence, and carbohydrate expression

We have analyzed the proteome of several strains of Haemophilus ducreyi by two-dimensional gel electrophoresis (2-DE) and mass spectrometry. Over 100 spots were analyzed from the soluble and insoluble protein fractions from the prototype strain 35000HP and 122 distinct proteins were identified. Functions of approximately 80% of the 122 proteins were deduced by identification with close homologues of Haemophilus influenzae. Four additional wild type and three mutant strains were also analyzed that vary in their virulence and/or outer-membrane lipooligosaccharide structures. Overall, the 2-DE gel maps of the wild type and mutant strains were similar to strain 35000HP, suggesting little proteome diversity in relation to carbohydrate expression and/or virulence. An exception was the Kenyan strain 33921 which contained significant differences in its proteome 2-DE map and also synthesizes an unusual LOS with a trisaccharide branch structure. This African strain may represent a prototype of a second clonal group of H. ducreyi.     

The Hd0053 gene of Haemophilus ducreyi encodes an alpha2,3-sialyltransferase

Haemophilus ducreyi is a Gram-negative bacterium that causes chancroid, a sexually transmitted genital ulcer disease. Different lipooligosaccharide (LOS) structures have been identified from H. ducreyi strain 35000, including those sialylated glycoforms. Surface LOS of H. ducreyi is considered an important virulence factor that is involved in ulcer formation, cell adhesion, and invasion of host tissue. Gene Hd0686 of H. ducreyi, designated lst (for lipooligosaccharide sialyltransferase), was identified to encode an alpha2,3-sialyltransferase that is important for the formation of sialylated LOS. Here, we show that Hd0053 of H. ducreyi genomic strain 35000HP, the third member of the glycosyltransferase family 80 (GT80), also encodes an alpha2,3-sialyltransferase that may be important for LOS sialylation.     

Use of the complete genome sequence information of Haemophilus influenzae strain Rd to investigate lipopolysaccharide biosynthesis

The availability of the complete 1.83-megabase-pair sequence of the Haemophilus influenzae strain Rd genome has facilitated significant progress in investigating the biology of H. influenzae lipopolysaccharide (LPS), a major virulence determinant of this human pathogen. By searching the H. influenzae genomic database, with sequences of known LPS biosynthetic genes from other organisms, we identified and then cloned 25 candidate LPS genes. Construction of mutant strains and characterization of the LPS by reactivity with monoclonal antibodies, PAGE fractionation patterns and electrospray mass spectrometry comparative analysis have confirmed a potential role in LPS biosynthesis for the majority of these candidate genes. Virulence studies in the infant rat have allowed us to estimate the minimal LPS structure required for intravascular dissemination. This study is one of the first to demonstrate the rapidity, economy and completeness with which novel biological information can be accessed once the complete genome sequence of an organism is available.     

Mutations in Haemophilus influenzae mismatch repair genes increase mutation rates of dinucleotide repeat tracts but not dinucleotide repeat-driven pilin phase variation rates

High-frequency, reversible switches in expression of surface antigens, referred to as phase variation (PV), are characteristic of Haemophilus influenzae. PV enables this bacterial species, an obligate commensal and pathogen of the human upper respiratory tract, to adapt to changes in the host environment. Phase-variable hemagglutinating pili are expressed by many H. influenzae isolates. PV involves alterations in the number of 5' TA repeats located between the -10 and -35 promoter elements of the overlapping, divergently orientated promoters of hifA and hifBCDE, whose products mediate biosynthesis and assembly of pili. Dinucleotide repeat tracts are destabilized by mismatch repair (MMR) mutations in Escherichia coli. The influence of mutations in MMR genes of H. influenzae strain Rd on dinucleotide repeat-mediated PV rates was investigated by using reporter constructs containing 20 5' AT repeats. Mutations in mutS, mutL, and mutH elevated rates approximately 30-fold, while rates in dam and uvrD mutants were increased 14- and 3-fold, respectively. PV rates of constructs containing 10 to 12 5' AT repeats were significantly elevated in mutS mutants of H. influenzae strains Rd and Eagan. An intact hif locus was found in 14 and 12% of representative nontypeable H. influenzae isolates associated with either otitis media or carriage, respectively. Nine or more tandem 5' TA repeats were present in the promoter region. Surprisingly, inactivation of mutS in two serotype b H. influenzae strains did not alter pilin PV rates. Thus, although functionally analogous to the E. coli MMR pathway and active on dinucleotide repeat tracts, defects in H. influenzae MMR do not affect 5' TA-mediated pilin PV.     

Stable shuttle vectors for Neisseria gonorrhoeae, Haemophilus spp. and other bacteria based on a single origin of replication

An origin of replication (ori) was obtained from a naturally occurring beta-lactamase-producing plasmid isolated from Neisseria gonorrhoeae and used to construct shuttle vectors capable of replicating in N. gonorrhoeae, Haemophilus ducreyi, Haemophilus influenzae and Escherichia coli. Using the gonococcal proAB genes, we complemented proline-requiring N. gonorrhoeae F62 and E. coli HB101 in trans. The first demonstration of the expression of the green fluorescent protein (GFP) in either N. gonorrhoeae or H. ducreyi was shown using this vector, indicating that GFP may be a useful tool in the analysis of these organisms. This is the first report of a gonococcal vector based on a broad host range, genetically defined ori, and should facilitate the molecular analysis of gonococcal and Haemophilus genes.     

An isogenic haemolysin-deficient mutant of Haemophilus ducreyi lacks the ability to produce cytopathic effects on human foreskin fibroblasts

The haemolysin of Haemophilus ducreyi is the newest member of the Proteus/Serratia family of pore-forming toxins. In order to assess the role of the haemolysin in virulence, we constructed an isogenic haemolysin-deficient mutant of H. ducreyi strain 35000. This strain, designated 35000-3, lacks detectable haemolytic activity. We tested H. ducreyi strains 35000 and 35000-3 for their cytopathic activity against human foreskin fibroblasts (HFFs). We observed strong cytopathic activity when strain 35000 was co-cultured with HFFs. In contrast, cytopathic activity was not observed when strain 35000-3 was co-cultured with HFF cells. We also analysed the isogenic pair of H. ducreyi strains for cytopathic activity against HeLa cells and the keratinocyte cell line HaCaT. Strains 35000 and 35000-3 were strongly cytotoxic when co-cultured with HeLa cells. HaCaT monolayers were slightly damaged by cocultivation with strain 35000-3 but this damage was much less than that observed when HaCaT cells were cocultured with strain 35000. These results indicate that the H. ducreyi haemolysin is responsible for the previously observed cytotoxic activity against HFF cells and is partially responsible for the activity observed with HaCaT cells. The haemolysin, however, is not responsible for the activity observed with HeLa cells.     

Comparative analyses of two cryptic plasmids from Haemophilus somnus (Histophilus somni)

Haemophilus somnus is an opportunistic bacterial pathogen capable of causing pneumonia, septicemia, and other systemic infections in bovines. An H. somnus isolate from bovine abortion (strain 649) was found to carry a approximately 1.3 kb plasmid (pHS649) that contained partial homology to two previously sequenced Haemophilus/Histophilus plasmids by BLAST analyses. Sequence analysis of pHS649 identified a putative RepA protein with 48% similarity to the RepA protein of Escherichia coli plasmid pKL1. A approximately 5 kb plasmid (pHS129) from H. somnus preputial isolate 129Pt was also sequenced and found to encode two copies of a putative RepB protein. Whereas pHS649 stably replicated in E. coli DH5alpha, pHS129 did not. Genetic relatedness and possible replication mechanisms of these plasmids are described.     

Proposed second class of Haemophilus ducreyi strains show altered protein and lipooligosaccharide profiles

Haemophilus ducreyi is the etiologic agent of chancroid, a sexually transmitted genital ulcer disease. Previously we have shown that the protein profiles and lipooligosaccharide (LOS) structures from various strains of H. ducreyi are generally well conserved. Previous studies have demonstrated that at least one strain, 33921, has a variant protein profile and LOS structure. In this study, both the whole cell lysate and the membrane proteins from strain 33921 were further examined and compared to the prototypical strain 35000HP by 2-DE and by the 16-BAC (benzyldimethyl-n-hexadecylammonium chloride)/SDS-PAGE two-detergent system, respectively. These comparisons demonstrated that a number of the proteins that could be identified from both strains had altered positions on the gels, both in their apparent molecular weight and pI values. Strain 33921 has been suggested to be a member of a second class of strains, termed class II strains. In this study, the proteomic profiles and the LOS structures from the five potential class II strains were examined and found to be similar to strain 33921.     

Origin and Direction of Haemophilus Bacteriophage HP1 DNA Replication

Rapidly growing Haemophilus influenzae strain Rd bacteria were infected with bacteriophage HP1 and DNA extracts prepared at various times thereafter. A number of phage genes scattered along the entire phage genome were quantitatively assayed by transformation. The kinetics of activity increases of these genes suggests that phage HP1 DNA replication begins at a fixed origin about one-quarter from the right end and that it proceeds to the left.     

Introduction of transposon Tn916 DNA into Haemophilus influenzae and Haemophilus parainfluenzae.

Enterococcus (Streptococcus) faecalis transposon Tn916 was introduced into Haemophilus influenzae Rd and Haemophilus parainfluenzae by transformation and demonstrated to transpose efficiently. Haemophilus transformants resistant to tetracycline were observed at a frequency of approximately 3 x 10(2) to 5 x 10(3)/micrograms of either pAM120 (pGL101::Tn916) or pAM180 (pAM81::Tn916) plasmid DNAs, which are incapable of autonomous replication in this host. Restriction enzyme analysis and Southern blot hybridization revealed that (i) Tn916 integrates into many different sites in the H. influenzae and H. parainfluenzae genomes; (ii) only the 16.4-kilobase-pair Tn916 DNA integrates, and no vector DNA was detected; and (iii) the Tetr phenotype was stable in the absence of selective pressure. Second-generation Tn916 transformants occurred at the high frequency of chromosomal markers and retained their original chromosomal locations. Similar results were obtained with H. influenzae Rd BC200 rec-1 as the recipient strain, which suggests host rec functions are not required in Tn916 integrative transposition. Transposition with Tn916 is an important procedure for mutagenesis of Haemophilus species.     

Survival of nontypeable Haemophilus influenzae in macrophages

In this study we have investigated the ability of nonencapsulated, nontypeable Haemophilus influenzae, NT477 to survive in the J774 mouse macrophage-like cell line. Viable, intracellular nontypeable H. influenzae could still be recovered from macrophages 72 h after phagocytosis. In contrast, H. influenzae strain Rd, an avirulent, nonencapsulated variant of a serotype d strain, was killed within 24 h. These differences suggest that NT477, in comparison to Rd, possesses unique attributes that enable it to survive in macrophages for prolonged periods. To determine whether this trait is ubiquitous amongst nontypeable H. influenzae, 33 primary clinical isolates obtained from children with otitis media were screened for their ability to survive in macrophages. Of these isolates, 82% were able to persist in an intracellular environment for periods of at least 24 h. The number of viable organisms recovered at this time ranged from 2x10(4) to 50 colony-forming units per strain indicating that the extent to which nontypeable H. influenzae can resist macrophage-mediated killing varies between strains.     

The other mediator for adherence of Haemophilus influenzae organisms without involvement of fimbriae.

The number of the nontypeable Haemophilus influenzae (HiN) organisms that adhered to the primary mouse fetal lung cells was significantly more than type b Haemophilus influenzae (Hib) organisms. The average number of HiN organisms adherent to host cells was 2,291/100 host cells (range, 1,654-3,182), but that of Hib was markedly reduced to 147/100 host cells (range, 102-238). In this case, P value was less than 0.05 by using a paired Student t-test. The sonicated extract from HiN TMS11 organisms inhibited adherence of H. influenzae TMS11 organisms to monolayer at 76.3% and it inhibited adherence of Hib TMS24 organisms at 92.3%. This result indicates that a mediator existing on the surface of HiN organisms may be the same as that on type b organisms. The number of detected organisms in broncho and lung tissues 3 days after intranasal infection with HiN strains was significantly greater than that in infection with Hib strains. Therefore, in vitro adhesive capacity of H. influenzae organisms was correlative to infectivity by intranasal injection.     

Genetic Analysis of a Pyocin-Resistant Lipooligosaccharide (LOS) Mutant of Haemophilus ducreyi: Restoration of Full-Length LOS Restores Pyocin Sensitivity

DNA sequence and Southern blot analyses were used to determine the genetic defect of a Haemophilus ducreyi pyocin-resistant lipooligosaccharide (LOS) mutant, HD35000R. The region of the HD35000R chromosome containing the suspected mutation was amplified, and sequence analysis detected a 3,189-bp deletion. This deletion resulted in the loss of the entire waaQ gene, another open reading frame that encodes a putative homolog to a hypothetical protein (HI0461) of H. influenzae, the gene encoding an argininosuccinate synthase homolog, and a change in the 3' sequence of the lgtF gene. Southern blot analysis confirmed that no genomic rearrangements had occurred. Isogenic LOS mutants and the respective complemented mutants were evaluated for susceptibility to pyocin C. The mutants expressing truncated LOS were resistant to lysis by pyocin C, and complementation restored sensitivity to the pyocin. We conclude that HD35000R is defective in both glycosyltransferase genes and that pyocin resistance is due to truncation of the full-length LOS molecule.     

Identification and characterization of a cell envelope protein of Haemophilus influenzae contributing to phase variation in colony opacity and nasopharyngeal colonization

Haemophilus influenzae undergoes spontaneous phase variation in colony morphology. Organisms from transparent colonies efficiently colonize the nasopharynx in an infant rat model of H. influenzae carriage, whereas organisms from more opaque colonies are deficient at colonization. A genetic approach relying on the transformability of H. influenzae was used to identify a locus contributing to opacity variation. By screening a library of chomosomal DNA from an opaque variant of strain Rd, it was possible to isolate a single clone capable of transforming a transparent Rd host to a more opaque phenotype. A region containing two genes, designated oapA and oapB , was identified. The deduced amino acid sequence of oapB has similarity to a consensus sequence for bacterial lipoproteins. Genetically defined mutations in oapA were transformed into the transparent Rd to confirm that this gene is required for expression of the transparent colony phenotype. Although oapA lacks a signal sequence, gene fusions to phoA show that OapA is secreted in H. influenzae and undergoes phase variation in expression. Mutagenesis of oapA in strain Rd, and type b strain Eagan, resulted in loss of the ability to colonize the nasopharynx of infant rats. The type b mutant, however, was as virulent as its parent strain when inoculated intraperitoneally. This suggests that the contribution of OapA to pathogenesis is limited to events associated with colonization of the mucosal surface.     

Genomic sequence of an otitis media isolate of nontypeable Haemophilus influenzae: comparative study with H. influenzae serotype d, strain KW20

In 1995, the Institute for Genomic Research completed the genome sequence of a rough derivative of Haemophilus influenzae serotype d, strain KW20. Although extremely useful in understanding the basic biology of H. influenzae, these data have not provided significant insight into disease caused by nontypeable H. influenzae, as serotype d strains are not pathogens. In contrast, strains of nontypeable H. influenzae are the primary pathogens of chronic and recurrent otitis media in children. In addition, these organisms have an important role in acute otitis media in children as well as other respiratory diseases. Such strains must therefore contain a gene repertoire that differs from that of strain Rd. Elucidation of the differences between these genomes will thus provide insight into the pathogenic mechanisms of nontypeable H. influenzae. The genome of a representative nontypeable H. influenzae strain, 86-028NP, isolated from a patient with chronic otitis media was therefore sequenced and annotated. Despite large regions of synteny with the strain Rd genome, there are large rearrangements in strain 86-028NP's genome architecture relative to the strain Rd genome. A genomic island similar to an island originally identified in H. influenzae type b is present in the strain 86-028NP genome, while the mu-like phage present in the strain Rd genome is absent from the strain 86-028NP genome. Two hundred eighty open reading frames were identified in the strain 86-028NP genome that were absent from the strain Rd genome. These data provide new insight that complements and extends the ongoing analysis of nontypeable H. influenzae virulence determinants.     

Tetrameric repeat units associated with virulence factor phase variation in Haemophilus also occur in Netsseria spp. and Moraxella catarrhalis

Abstract The tetrameric repeat units 5'-CAAT-3' and 5'-GCAA-3' are associated with phase variable expression of lipopolysaccharide biosynthetic genes in Haemophilus influenzae . Four other tetrameric repeat units have also been reported from H. influenzae strain Rd, 5'-CAAC-3', 5'-GACA-3', 5'-AGCT-3', and 5'-TTTA-3', which are also associated with putative virulence factors. Using oligonucleotide probes corresponding to five tandem copies of each of these tetramers, we have screened three strains of Neisseria meningitidis and one each of Neisseria gonorrhoeae, Neisseria lactamica, Haemophilus parainfluenzae, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiceptica and Moraxella catarrhalis for the presence of these motifs. We have demonstrated the presence of multiple copies of the 5'-GCAA-3' motif in all the Neisseria strains tested, and also the repeated motif 5'-CAAC-3' in M. catarrhalis . We have further demonstrated by Southern blot analysis that the 5'-CAAC-3' repeats detected in M. catarrhalis are probably associated with the same genes as in H. influenzae , but that the 5'-GCAA-3' motifs in N. meningitidis are not. The use of characterised tetrameric DNA sequences as hybridisation probes may prove useful in the identification of novel phase variable virulence determinants in organisms other than H. influenzae .     

Distribution of diaminopropane, putrescine and cadaverine in Haemophilus and Actinobacillus

Cellular levels of diaminopropane, putrescine and cadaverine, and decarboxylase activities to produce these diamines in six species (16 strains) of Haemophilus and four species (5 strains) of Actinobacillus belonging to the family Pasteurellaceae of the gamma subclass of the class Proteobacteria, were determined by high performance liquid chromatography (HPLC). Diaminopropane was ubiquitously distributed within all Haemophilus and Actinobacillus species, and L-2,4-diaminobutyric acid decarboxylase activity was detected in them. Putrescine and ornithine decarboxylase activity were found in H. aphrophilus, H. parainfluenzae and H. influenzae (type a, b, d, e and f except for type c) but not detected in H. aegyptius, H. parahaemolyticus, H. ducreyi and Actinobacillus species. Cadaverine occurred in H. aphrophilus, H. aegyptius, H. influenzae, H. parainfluenzae, A. actinomycetemcomitans, A. equuli and A. lignieresii, whereas their lysine decarboxylase activity was scarcely detected. Cadaverine was not found in H. parahaemolyticus, H. ducreyi and A. suis. The diamine profile serves as a phenotypic marker for the chemotaxonomic classification of the family Pasteurellaceae.