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.     

Complete genome sequence of Haemophilus somnus (Histophilus somni) strain 129Pt and comparison to Haemophilus ducreyi 35000HP and Haemophilus influenzae Rd

Haemophilus somnus can be either a commensal of bovine mucosal surfaces or an opportunistic pathogen. Pathogenic strains of H. somnus are a significant cause of systemic disease in cattle. We report the genome sequence of H. somnus 129Pt, a nonpathogenic commensal preputial isolate, and the results of a genome-wide comparative analysis of H. somnus 129Pt, Haemophilus influenzae Rd, and Haemophilus ducreyi 35000HP. We found unique genes in H. somnus 129Pt involved in lipooligosaccharide biosynthesis, carbohydrate uptake and metabolism, cation transport, amino acid metabolism, ubiquinone and menaquinone biosynthesis, cell surface adhesion, biosynthesis of cofactors, energy metabolism, and electron transport. There were also many genes in common among the three organisms. Our comparative analyses of H. somnus 129Pt, H. influenzae Rd, and H. ducreyi 35000HP revealed similarities and differences in the numbers and compositions of genes involved in metabolism, host colonization, and persistence. These results lay a foundation for research on the host specificities and niche preferences of these organisms. Future comparisons between H. somnus 129Pt and virulent strains will aid in the development of protective strategies and vaccines to protect cattle against H. somnus disease.     

Evolution of an Autotransporter: Domain Shuffling and Lateral Transfer from Pathogenic Haemophilus to Neisseria

The genomes of pathogenic Haemophilus influenzae strains are larger than that of Rd KW20 (Rd), the nonpathogenic laboratory strain whose genome has been sequenced. To identify potential virulence genes, we examined genes possessed by Int1, an invasive nonencapsulated isolate from a meningitis patient, but absent from Rd. Int1 was found to have a novel gene termed lav, predicted to encode a member of the AIDA-I/VirG/PerT family of virulence-associated autotransporters (ATs). Associated with lav are multiple repeats of the tetranucleotide GCAA, implicated in translational phase variation of surface molecules. Laterally acquired by H. influenzae, lav is restricted in distribution to a few pathogenic strains, including H. influenzae biotype aegyptius and Brazilian purpuric fever isolates. The DNA sequence of lav is surprisingly similar to that of a gene previously described for Neisseria meningitidis. Sequence comparisons suggest that lav was transferred relatively recently from Haemophilus to Neisseria, shortly before the divergence of N. meningitidis and Neisseria gonorrhoeae. Segments of lav predicted to encode passenger and beta-domains differ sharply in G+C base content, supporting the idea that AT genes have evolved by fusing domains which originated in different genomes. Homology and base sequence comparisons suggest that a novel biotype aegyptius AT arose by swapping an unrelated sequence for the passenger domain of lav. The unusually mobile lav locus joins a growing list of genes transferred from H. influenzae to Neisseria. Frequent gene exchange suggests a common pool of hypervariable contingency genes and may help to explain the origin of invasiveness in certain respiratory pathogens.     

A clonal group of nontypeable Haemophilus influenzae with two IgA proteases is adapted to infection in chronic obstructive pulmonary disease

Strains of nontypeable Haemophilus influenzae show enormous genetic heterogeneity and display differential virulence potential in different clinical settings. The igaB gene, which encodes a newly identified IgA protease, is more likely to be present in the genome of COPD strains of H. influenzae than in otitis media strains. Analysis of igaB and surrounding sequences in the present study showed that H. influenzae likely acquired igaB from Neisseria meningitidis and that the acquisition was accompanied by a ~20 kb genomic inversion that is present only in strains that have igaB. As part of a long running prospective study of COPD, molecular typing of H. influenzae strains identified a clonally related group of strains, a surprising observation given the genetic heterogeneity that characterizes strains of nontypeable H. influenzae. Analysis of strains by 5 independent methods (polyacrylamide gel electrophoresis, multilocus sequence typing, igaB gene sequences, P2 gene sequences, pulsed field gel electrophoresis) established the clonal relationship among the strains. Analysis of 134 independent strains collected prospectively from a cohort of adults with COPD demonstrated that ~10% belonged to the clonal group. We conclude that a clonally related group of strains of nontypeable H. influenzae that has two IgA1 protease genes (iga and igaB) is adapted for colonization and infection in COPD. This observation has important implications in understanding population dynamics of H. influenzae in human infection and in understanding virulence mechanisms specifically in the setting of COPD.     

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.     

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.     

Characterization of the IgD binding site of encapsulated Haemophilus influenzae serotype b

Encapsulated Haemophilus influenzae is a causative agent of invasive disease, such as meningitis and septicemia. Several interactions exist between H. influenzae and the human host. H. influenzae has been reported to bind IgD in a nonimmune manner, but the responsible protein has not yet been identified. To define the binding site on IgD for H. influenzae, full-length IgD and four chimeric IgDs with interspersed IgG sequences and Ag specificity for dansyl chloride were expressed in stably transfected Chinese hamster ovary cells. The binding of recombinant IgD to a panel of encapsulated H. influenzae serotype b (Hib) and nontypeable strains were investigated using a whole cell ELISA and flow cytometry. IgD binding was detected in 50% of the encapsulated Hib strains examined, whereas nontypeable H. influenzae did not interact with IgD. Finally, mapping experiments using the chimeric IgD/IgG indicated that IgD CH1 aa 198-224 were involved in the interaction between IgD and H. influenzae. Thus, by using recombinant IgD and chimeras with defined Ag specificity, we have confirmed that Hib specifically binds IgD, and that this binding involves the IgD CH1 region.     

Mechanism of Haemophilus influenzae transfection by single and double prophage deoxyribonucleic acid.

Whole phages HP1 and HP3, vegetative-phage deoxyribonucleic acid (DNA), and single and tandem double prophage DNA were exposed to ultraviolet radiation and then assayed on a wild-type (DNA repair-proficient) Haemophilus influenzae Rd strain and on a repair-deficient uvr-1 strain. Host cell reactivation (DNA repair) was observed for whole-phage and vegetative-phage DNA but not for single and double prophage DNA. Competent (phage-resistant) Haemophilus parainfluenzae cells were normally transfected with H. influenzae-grown phage DNA and with tandem double prophage DNA but not at all with single prophage DNA. CaCl2-treated H. influenzae suspensions could be transfected with vegetative phage DNA and with double prophage DNA but not with single prophage DNA. These observations support the hypothesis that transfection with single prophage DNA occurs through prophage DNA single-strand insertion into the recipient chromosome (at the bacterial att site) followed by DNA replication and then prophage induction.     

Duplication of pilus gene complexes of Haemophilus influenzae biogroup aegyptius.

Brazilian purpuric fever (BPF) is a recently described pediatric septicemia caused by a strain of Haemophilus influenzae biogroup aegyptius. The pilus specified by this bacterium may be important in BPF pathogenesis, enhancing attachment to host tissue. Here, we report the cloning of two haf (for H. influenzae biogroup aegyptius fimbriae) gene clusters from a cosmid library of strain F3031. We sequenced a 6.8-kb segment of the haf1 cluster and identified five genes (hafA to hafE). The predicted protein products, HafA to HafD, are 72, 95, 98, and 90% similar, respectively, to HifA to HifD of the closely related H. influenzae type b pilus. Strikingly, the putative pilus adhesion, HifE, shares only 44% identity with HafE, suggesting that the proteins may differ in receptor specificity. Insertion of a mini-gammadelta transposon in the hafE gene eliminated hemadsorption. The nucleotide sequences of the haf1 and haf2 clusters are more than 99% identical. Using the recently published sequence of the H. influenzae Rd genome, we determined that the haf1 complex lies at a unique position in the chromosome between the pmbA gene and a hypothetical open reading frame, HI1153. The location of the haf2 cluster, inserted between the purE and pepN genes, is analogous to the hif genes on H. influenzae type b. BPF fimbrial phase switching appears to involve slip-strand mispairing of repeated dinucleotides in the pilus promoter. The BPF-associated H. influenzae biogroup aegyptius pilus system generally resembles other H. influenzae, but the possession of a second fimbrial gene cluster, which appears to have arisen by a recent duplication event, and the novel sequence of the HafE adhesin may be significant in the unusual pathogenesis of BPF.     

Study on genetic diversity of genes encoding main adhesins among nontypeable Haemophilus influenzae strains isolated from children in Poland

The study was based on hypothesis that in the nontypeable population of H. influenzae strains isolated from children there are some genetically predisposed to induce symptomatic infection in children and that they might be divided into different groups depending on profiles of genes encoding main adhesins synthesis. The work aimed at analysis of distribution of genes encoding adhesins and evaluation of domination possibility of some strains representing particular adhesins genes profiles among NTHi population. Results of the study revealed that among population of NTHi strains, distribution of genes encoding main adhesins are differing. Among children, NTHi strains harbouring genes encoding HA and HMW1/HMW2 adhesins were more prevalent in healthy children and in children with symptomatic infections, respectively. Analysis of strains harbouring main adhesins profiles might be a useful screening method in monitoring strains circulating among children, in order to determine the most invasive NTHi strains.     

The identification of the bacteriophage HP1c1 and S2 integration sites in Haemophilus influenzae Rd by field-inversion gel electrophoresis of large DNA fragments.

The resolution of high molecular weight DNA fragments by field-inversion gel electrophoresis (FIGE) demonstrate the presence of two phage (S2 and HP1c1) integration sites (attB) in the Haemophilus influenzae Rd chromosome. In a population of wild-type cells either prophage site appears to be occupied in a single cell by one to at least three, tandemly repeated, amplified phage DNA molecules. The attL of the second bacterial attachment site present in the host SmaI fragment 7 and the leftmost part of phage S2 type B DNA of its genome organization (Piekarowicz et. al., 1986) have been sequenced. A comparison of the two bacterial att sites demonstrated that their homology is limited to the core region. A comparison of the DNA sequences of phage S2 type B and HP1c1 type C revealed a 530-bp insertion in the HP1c1 type C (not present in S2 type B) in addition to DNA variants due mostly to single-base mismatches. We postulate that phage S2 and HP1c1 genome variants (A, B, and C) evolved from a single phage origin and might stem from passage history arisen through accumulation of mutations.     

Fragmentation heterogeneity of 23S ribosomal RNA in Haemophilus species

The fragmentation of 23S rRNA of 22 Haemophilus influenzae strains and eight strains belonging to other Haemophilus species was investigated. Instead of intact molecules, the 23S rRNA molecules were found to be cleaved into two to five smaller conserved fragments in most strains examined, especially in H. influenzae type b (5/6) and nontypeable strains (5/5). One or two conserved potential cleavage sites were identified by PCR analysis of the strains showing a fragmented 23S rRNA pattern. The relevant nucleotide sequences were determined and compared to H. influenzae Rd, which contains intact 23S rRNA molecules. An identical 112 bp long intervening sequence (IVS) at position 542 and a conserved 121–123 bp IVS sequence at position 1171 were found in two H. influenzae type b strains and one nontypeable strain. Among the strains with fragmented 23S rRNA, nearly half showed a heterogeneous cleavage pattern due to the dispersion of IVSs among different 23S rRNA operons. The localization of the conserved H. influenzae IVSs coincided well with the extensively studied IVSs among other bacteria, but differed in nucleotide sequence from any other reported IVSs. Therefore, the IVSs of Haemophilus 23S rRNA may originate from a common source that is independent of other bacteria.     

流感嗜血杆菌的分型研究

目的分析同济医院分离的流感嗜血杆菌的生物学分型及荚膜基因分型,了解本地区分离的流感嗜血杆菌的主要流行株。方法2012年1月1日至2012年12月31日从华中科技大学同济医学院附属同济医院分离流感嗜血杆菌100株。根据脲酶、吲哚和鸟氨酸脱羧酶试验对流感嗜血杆菌进行传统的生物学分型,分为Ⅰ～Ⅷ八个生物型。回顾患者病史资料,分析生物学分型和流感嗜血杆菌所引起的疾病之间的关系。用流感嗜血杆菌荚膜编码基因（bexA）和a—f型特异性荚膜基因设计引物,采用PCR技术对流感嗜血杆菌进行荚膜基因检测。通过生物学分型和荚膜基因分型结果的比对,探讨两者之间的关联。结果分离的100株流感嗜血杆菌生物学分型结果如下：Ⅲ型6株,Ⅳ型28株,Ⅴ型1株,Ⅵ型54株,Ⅶ型11株。未分离到Ⅰ型、Ⅱ型和Ⅷ型。分析患者的临床诊断,发现主要流行株Ⅵ型流感嗜血杆菌主要引起患者肺炎（包括支气管肺炎和新生儿肺炎）和支气管炎（包括毛细支气管炎和喘息性支气管炎）。荚膜基因分型结果显示,未分离到b型和b-型流感嗜血杆菌。共分离到1株f型,其余99株均为无荚膜抗原的不可分型流感嗜血杆菌。生物学分型和荚膜分型之间无明显的相关性。结论该院分离的流感嗜血杆菌主要为生物型Ⅵ型。回顾患者病史,发现Ⅵ型主要引起肺炎和支气管炎。荚膜基因分型显示,本地区分离的流感嗜血杆菌主要为不可分型流感嗜血杆菌。生物学分型和荚膜基因分型之间无明显相关性。     

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.     

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.     

Structural profiling of lipopolysaccharide glycoforms expressed by non-typeable Haemophilus influenzae: phenotypic similarities between NTHi strain 162 and the genome strain Rd

Non-typeable Haemophilus influenzae (NTHi) is a significant cause of otitis media in children. We have employed single and multiple step electrospray ionization mass spectrometry (ESIMS) and NMR spectroscopy to profile and elucidate lipopolysaccharide (LPS) structural types expressed by NTHi strain 162, a strain obtained from an epidemiological study in Finland. ESIMS on O-deacylated LPS (LPS-OH) and core oligosaccharide (OS) samples of LPS provided information on the composition and relative abundance of glycoforms differing in the number of hexoses linked to the conserved inner-core element, L-alpha-D-Hepp-(1-->2)-[PEtn-->6]-L-alpha-D-Hepp-(1-->3)-L-alpha-D-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdop-(2-->6)-Lipid A of H. influenzae LPS. The strain examined was found to elaborate Hex2 to Hex5 LPS glycoform populations having structures identical to those observed for H. influenzae strain Rd [Risberg, A.; Masoud, H.; Martin, A.; Richards, J.C.; Moxon, E.R.; Schweda, E.K.H. Eur. J. Biochem. 1999, 261, 171-180], the strain for which the complete genome has been sequenced. In addition, sialyllactose-containing glycoforms previously identified in strain Rd as well as several NTHi strains, were identified as minor components. Multiple step tandem ESIMS (MS(n)) on dephosphorylated and permethylated OS provided information on the arrangement of glycoses within the major population of glycoforms and on the existence of additional isomeric glycoforms. Minor Hex1 and Hex6 glycoforms were detected and characterized where the Hex6 glycoform was comprised of a dihexosamine-containing pentasaccharide chain attached at the proximal heptose residue of the inner-core unit. LPS structural motifs present in the NTHi strain 162 are expressed by a genetically diverse set of disease causing isolates, providing the basis for a vaccine strategy against NTHi otitis media.     

The length of a tetranucleotide repeat tract in Haemophilus influenzae determines the phase variation rate of a gene with homology to type III DNA methyltransferases

Haemophilus influenzae is an obligate commensal of the upper respiratory tract of humans that uses simple repeats (microsatellites) to alter gene expression. The mod gene of H. influenzae strain Rd has homology to DNA methyltransferases of type III restriction/modification systems and has 40 tetranucleotide (5'-AGTC) repeats within its open reading frame. This gene was found in 21 out of 23 genetically distinct H. influenzae strains, and in 13 of these strains the locus contained repeats. H. influenzae strains were constructed in which a lacZ reporter was fused to a chromosomal copy of mod downstream of the repeats. Phase variation occurred at a high frequency in strains with the wild-type number of repeats. Mutation rates were derived for similarly engineered strains, containing different numbers of repeats. Rates increased linearly with tract length over the range 17-38 repeat units. The majority of tract alterations were insertions or deletions of one repeat unit with a 2:1 bias towards contractions of the tract. These results demonstrate the number of repeats to be an important determinant of phase variation rate in H. influenzae for a gene containing a microsatellite.     

Cloning and expression of the Haemophilus influenzae transferrin receptor genes

The genomic transferrin receptor genes ( tbpA and tbpB  ) from two strains of Haemophilus influenzae type b (Hib) and two strains of non-typable H. influenzae (NTHi) have been cloned and sequenced. The deduced protein sequences of the H. influenzae tbpA genes were 95–100% conserved and those of the tbpB genes were 66–100% conserved. The tbpB gene from one strain of NTHi was found to encode a truncated Tbp2 protein. The tbpB genes from four additional NTHi strains were amplified by the polymerase chain reaction (PCR) utilizing primers derived from the conserved N-terminal sequences of Tbp1 and Tbp2 and were found to encode full-length proteins. Although several bacterial species express transferrin receptors, when the Tbp1 and Tbp2 sequences from different organisms were compared, there was only limited homology. Recombinant Tbp1 and Tbp2 proteins were expressed from Escherichia coli and antisera were raised to the purified proteins. There was significant antigenic conservation of both Tbp1 and Tbp2 amongst H. influenzae strains, as determined by Western blot analysis. In a passive model of bacteraemia, infant rats were protected from challenge with Hib after transfer of anti-rTbp2 antiserum, but not after anti-rTbp1 antiserum.