Phase variation of Haemophilus influenzae lipopolysaccharide: Characterization of lipopolysaccharide from individual colonies

Abstract The lipopolysaccharide (LPS) of Haemophilus influenzae expresses a number of core oligosaccharide epitopes on its outer surface. The expression of individual epitopes is subject to frequent (approximately 1% bacteria/generation) reversible phase variation, as determined by colony immunoblots. We have used a microtechnique for the extraction of LPS from individual colonies, whose LPS antigenic phenotype has been identified, so that the LPS can be studied by tricine sodium dodecylsulphate polyacrylamide gel electrophoresis (T-SDS-PAGE). This avoids the introduction of heterogenous phase-varying LPS which is inevitable if bacteria from colonies are grown in broth culture prior to LPS extraction and analysis. Using these techniques we have investigated the repertoire of LPS phase variation exhibited by H. influenzae strain RM7004 (a serotype b meningitis isolate). This technique will facilitate the study of bacteria in which there is variable LPS expression.     

RecJ, ExoI and RecG are required for genome maintenance but not for generation of genetic diversity by repeat-mediated phase variation in Haemophilus influenzae

High levels of genetic diversity are generated in Haemophilus influenzae populations through DNA repeat-mediated phase variation and recombination with DNA fragments acquired by uptake from the external milieu. Conversely, multiple pathways for maintenance of the genome sequence are encoded in H. influenzae genomes. In Escherichia coli, mutations in single-stranded-DNA exonucleases destabilise tandem DNA repeats whilst inactivation of recG can stabilise repeat tracts. These enzymes also have varying effects on recombination. Deletion mutations were constructed in H. influenzae genes encoding homologs of ExoI, RecJ and RecG whilst ExoVII was refractory to mutation. Inactivation of RecJ and RecG, but not ExoI, increased sensitivity to irradiation with ultraviolet light. An increase in spontaneous mutation rate was not observed in single mutants but only when both RecJ and ExoI were mutated. None of the single- or double-mutations increased or decreased the rates of slippage in tetranucleotide repeat tracts. Furthermore, the exonuclease mutants did not exhibit significant defects in horizontal gene transfer. We conclude that RecJ, ExoI and RecG are required for maintenance of the H. influenzae genome but none of these enzymes influence the generation of genetic diversity through mutations in the tetranucleotide repeat tracts of this species.     

Population genetics and antibiotic susceptibility of invasive Haemophilus influenzae in Manitoba, Canada, from 2000 to 2006

One hundred and twenty-two isolates of Haemophilus influenzae causing invasive disease were collected in Manitoba, Canada, from 2000 to 2006 and examined for serotype, biotype, sequence type (ST) by multilocus sequence typing and antibiotic susceptibility. Nonserotypeable (NST) isolates accounted for over half of the isolates collected (69 isolates, 56.6%). There were 36 serotype a, five serotype b, two serotype c, one serotype d, four serotype e and five serotype f isolates collected. The 69 NST isolates were found to be very diverse, with isolates representing six biotypes and 45 STs. The serotypeable isolates were more clonal, with each of the serotypes showing little diversity in their biotypes and STs. Of the 122 isolates, 17% were resistant to ampicillin due to beta-lactamase production, 10.7% were resistant to trimethoprim-sulfamethoxazole, 1.6% were resistant to clarithromycin, 2.5% were resistant to amoxicillin-clavulanic acid and none was resistant to ciprofloxacin or moxifloxacin. Antibiotic resistance was more common in the NST strains, with 37.7% showing resistance to at least one antibiotic compared to 15% in the serotypeable strains. The results of this study suggest a shift in the epidemiology of invasive H. influenzae infections in the post-Hib vaccine era, and surveillance should include all serotypeable and NST isolates.     

Epidemiology of Haemophilus influenzae type b invasive disease in Wales.

OBJECTIVE--To investigate the epidemiology of invasive disease due to Haemophilus influenzae type b, the clones responsible, and the antibiotic resistance of the isolates. DESIGN--Prospective population based analysis of clinical and epidemiological data collected for Gwynedd during 1980-90 and in the whole of Wales during 1988-90. SETTING--19 hospitals in Wales; all medical microbiology laboratories in Wales participated. PATIENTS--82 patients with confirmed invasive infections caused by H influenzae type b in Gwynedd during 1980-90 and 207 in Wales during 1988-90. MAIN OUTCOME MEASURES--Clinical and epidemiological measures; analysis of the clonal types of the isolates based on the electrophoretic mobilities of 17 metabolic enzymes; and antibiotic resistance. RESULTS--The annual incidence of H influenzae type b infections in Gwynedd was 3.2 cases/100,000 and in Wales was 2.5 cases/100,000. Most cases occurred in children aged under 5 years, the highest annual incidence being in those aged under 1 (84.6/100,000 and 56.9/100,000 in Wales). The cumulative risk of acquiring H influenzae type b disease by the fifth birthday was one in 456 in Gwynedd and one in 578 in Wales. Fifteen per cent of cases in Gwynedd and 7% of those in Wales occurred in adults. Predominant clinical conditions were meningitis in children and pneumonia in adults. In Gwynedd 2/70 (3%) children and 5/12 (42%) adults died. Long term neurological sequelae occurred in 8% (4/48) of children who survived haemophilus meningitis. Children presenting with infection were usually the youngest members of their family. No secondary household cases were identified. 100 of 128 (78%) strains were of a single clone, electrophoretic type 12.5, and 4/207 (1.9%) isolates from Wales were resistant to both ampicillin and chloramphenicol. CONCLUSIONS--The annual rate of infection in children aged under 5 in four Welsh counties was 12-44% higher than that previously published for the United Kingdom. The study emphasises the potential value of a vaccine effective in early infancy and provides baseline data to assess its efficacy after its introduction. Alternatives to ampicillin and chloramphenicol should be used as first line, empirical treatment for severe infections that might be caused by H influenzae type b in Wales.     

Molecular determinants of the pathogenesis of disease due to non-typable Haemophilus influenzae

Non-typable Haemophilus influenzae is a common commensal organism in the human upper respiratory tract and an important cause of localized respiratory tract disease. The pathogenesis of disease begins with bacterial colonization of the nasopharynx, a process that involves establishment on the mucosal surface and evasion of local immune mechanisms. Under the proper circumstances, the organism spreads contiguously to the middle ear, the sinuses, or the lungs, and then stimulates a brisk inflammatory response, producing symptomatic infection. In this review, we summarize our present understanding of the molecular determinants of this sequence of events. Continued investigation of the molecular mechanism of non-typable H. influenzae pathogenicity should facilitate development of novel approaches to the treatment and prevention of H. influenzae disease.     

In vitro cytotoxicity of Haemophilus influenzae lipopolysaccharides for bovine aortal endothelial cells

The cytotoxicity of purified lipopolysaccharide (LPS) from a prototype Haemophilus influenzae type b (Hib) strain (Eagan) and three transformants, differing in their LPS phenotype, for bovine aortal endothelial cells (BAOEC) was investigated. All LPS preparations caused cell disruption and release of lactate dehydrogenase (LDH), an indicator of cytotoxicity, from BAOEC monolayers but to differing extents. There was no correlation between the cytotoxicity of purified Hib LPS to BAOEC monolayers and potential to cause bacteraemia in experimental animals.     

Chemical evidence for covalent linkages of a semisynthetic glycoconjugate vaccine forHaemophilus influenzae type b disease

We have defined the nature of the covalent linkages in aHaemophilus influenzae type b oligosaccharide-CRM₁₉₇ conjugate vaccine, designated HbOC. The conjugate was acid hydrolyzed to release a novel amino-acid derivative,N-(2-hydroxyethyl)lysine (OHEt-Lys), identifiable with an amino-acid analyzer. This amino-acid derivative was formed by reduction of Schiff bases formed betweenH. influenzae type b oligosaccharides (HbO) and the lysyl -amino groups of CRM₁₉₇ (a non-toxic, cross-reactive variant of diphtheria toxin), followed by acid hydrolysis of HbOC. Quantification of OHEt-Lys per CRM₁₉₇ molecule allowed the determination of a covalency ratio, a useful parameter for evaluating the stoichiometry and consistency of HbOC preparations. Covalent association between HbO and CRM₁₉₇ was also demonstrated by the coincidence of immunoreactivity of gelelectrophoresed HbOC on a Western blot probed with anti-CRM₁₉₇ and anti-saccharide antisera.     

The identification a novel gene required for lipopolysaccharide biosynthesis by Haemophilus influenzae RM7004, using transposon Tn916 mutagenesis

Abstract Mutagenesis with the transposon Tn916 was used as a strategy to identify genes required for synthesis of the Galα(1–4)βGal component of Haemophilus influenzae strain RM7004 lipopolysaccharide. Insertion of Tn916 into an open reading frame (ORF) encoding a protein with 75% homology to the Escherichia coli methionine related protein (Mrp) is described. Mutations in mrp resulted in loss of reactivity with monoclonal antibody (mAb) 4C4, which recognises Galα(1–4)βGal, and expression of LPS with a different electrophoretic profile to that of wild-type RM7004. An unexpected feature of this mutation was that it appeared to influence the number of copies of 5'-CAAT-3' present in lic2A , a gene which is also required for biosynthesis and phase variable expression of the Galα(1–4)βGal LPS epitope.     

Presence of multiple copies of capsulation loci in invasive Haemophilus influenzae type b (Hib) strains in Japan before introduction of the Hib conjugate vaccine

Despite the effectiveness of the Hib vaccine, multiple amplification of the capb locus contributes to vaccine failure. However, there has been no report on the effect of Hib locus amplification in Japan. We examined 24 Hib strains from Japanese children with invasive diseases due to Hib. Although all strains showed the same capb sequence, Southern blot analysis showed that four strains (16.7%) harbored multiple copies (more than two) of the capb locus. Careful analysis of the locus in circulating Hib strains is necessary now that the Hib vaccine has been introduced into Japan.     

The heme-binding lipoprotein (HbpA) of Haemophilus influenzae: role in heme utilization

Haemophilus influenzae has an absolute growth requirement for heme and a heme binding lipoprotein (HbpA) has been implicated in the utilization of this essential nutrient. HbpA was identified by examining clones from an H. influenzae genomic library that caused Escherichia coli harboring the clone to bind heme. However, HbpA has not been shown to mediate heme acquisition in H. influenzae. We constructed an insertional mutation of hbpA in a nontypeable H. influenzae strain and demonstrated a role for the gene in utilization of multiple heme sources. This is the first report confirming a role for HbpA in utilization of heme.     

ELISA方法测定血清中Hib多糖抗体含量的条件比较研究

分别以牛血清白蛋白和人血清白蛋白作为封闭液的主要成分测定7份血清中抗b型流感嗜血杆菌荚膜多糖的抗体含量,结果显示,两组数据之间没有显著性差异(P>0.05)。分别用HRP标记的羊抗人IgG和HRP标记的鼠抗人IgG测定该7份血清,结果显示两组结果无显著性差异(P>0.05)。同时,将血清反应的温度和时间由37℃1.5h改为4℃16h(过夜),结果显示两者无显著性差异(P>0.05)。从而优化了该方法。     

流感嗜血杆菌的基因组学研究进展

流感嗜血杆菌Rd型是第一个被测序的细菌.其基因组大小为1830137bp,A含量为31%,C为19%,G为19%,T为31%,GC含量为38%,TIGR确认了1473个具有重要功能的基因.基因组序列中包含有复制子、核糖体启动子、毒力基因、DNA转运系统、调节子等结构.流感嗜血杆菌的基因组研究必将对寻找疾病相关基因和抗原基因,筛选药物作用靶位,研制特异的诊断试剂、药物和疫苗具有十分重要的科学意义.对流感嗜血杆菌的基因组学研究进展进行了综述.     

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.     

Influence of iron restriction on growth and the expression of outer membrane proteins by Haemophilus influenzae and H. parainfluenzae

Abstract The growth of both Haemophilus influenzae and H. parainfluenzae was progressively inhibited in media containing increasing concentrations of the iron chelator desferrioxamine. Iron restriction had little effect on the outer-membrane (OM) protein profiles of type b or non-typable H. influenzae although replacement of haemin with protoporphyrin IX resulted in the induction of an M _r 73 000 protein in the type b strains. H. parainfluenzae , however, responded to iron restriction by inducing several new proteins in the range of M _r 70 000 to 86 000.     

Siderophore-independent acquisition of transferrin-bound iron by Haemophilus influenzae type b

The specificity by which Haemophilus species acquired iron from transferrin (TF) was investigated. In a plate bioassay H. influenzae used iron bound to human, bovine and rabbit TFs but not mouse, rat, dog, horse, guinea-pig, pig or ovo- TFs or human and bovine lactoferrins. In contrast, H. pleuropneumoniae used iron only from pig TF whilst H. parainfluenzae was unable to utilize iron bound to any of the human or animal TFs tested. The inhibition of growth imposed on H. influenzae type b strain Eagan by the addition of the synthetic iron chelator EDDA to the culture medium was reversed by 30% iron-saturated human TF added directly to the medium but not when the TF was contained inside a dialysis bag. Dot-blotting of whole cells revealed that human TF bound to the surface of bacteria cultured in iron-restricted but not in iron-plentiful media. Incubation of whole bacterial cells in the presence of the proteolytic enzyme trypsin also abolished TF-binding activity, suggesting that the TF receptor was a protein. In competition dot blotting experiments, human and bovine but not rabbit, dog, mouse or guinea-pig TFs blocked the binding of a horseradish peroxidase--human TF conjugate. SDS-PAGE and Western blotting of outer membranes revealed the presence of a TF-binding protein of approximately 72 kDa. These results suggest that the acquisition of TF-bound iron by H. influenzae type b probably involves a direct interaction with an outer-membrane protein which shows some TF-species specificity.     

The capsular polysaccharide of Haemophilus influenzae type B prevents killing by complement and antibodies against outer membrane protein a

Abstract The ability of antibodies, raised in rabbits against purified outer membrane protein a ( M _r 47 000) of Haemophilus influenzae type b, to promote complement-dependent killing of these encapsulated organisms was investigated. Killing of encapsulated strains was not induced by these antibodies in conjunction with either human, mouse, rabbit or guinea-pig complement. Acapsular mutants were effectively killed by complement in the presence of antibodies against protein a . Killing was dependent on the presence of the 47-kDa protein a and was not influenced by the outer membrane protein subtype or lipopolysaccharide serotype of the strain. The killing-promoting activity could be absorbed from the sera with cells of strains with the same protein a , purified protein a , but not by purified lipopolysaccharide and capsular polysaccharide. Binding experiments showed that the encapsulated strain and its acapsular mutant bound antibodies against protein a with the same rate and to the same extent, indicating that the capsule probably interferes with complement activation or insertion of the membrane attack complex into the bacterial cell.