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.     

Carcinoembryonic antigens are targeted by diverse strains of typable and non-typable Haemophilus influenzae

Haemophilus influenzae (Hi), a commensal of the human respiratory mucosa, is an important cause of localized and systemic infections. We show that distinct strains belonging to typable (THi) and non-typable (NTHi) H. influenzae target human carcinoembryonic antigens (the membrane associated CEA family of cell adhesion molecules, are now termed CEACAMs). All strains of H. influenzae biogroup aegyptius (Hi-aeg) and more than 70% of THi and NTHi strains tested specifically recognize CEACAMI-Fc soluble constructs. Furthermore, transfection of Chinese hamster ovary cells with human CEACAM1 cDNA alone was sufficient for promoting Hi interactions with the transfected cells. The majority of the Hi-aeg strains tested interacted with soluble constructs containing only the N-terminal domain. In contrast, several THi and NTHi strains reacted with soluble constructs only when additional extracellular A and B domains of the receptor were present. The use of monoclonal antibodies confirmed that THi and NTHi strains also interact primarily at the N-domain. We used site-directed mutants of CEACAM1 that contained substitutions at surface exposed amino acids and a molecular model of the N-domain to identify the residues involved in interactions with Hi ligands. The studies show that a common region exposed at the CFG face of the molecule is targeted by diverse Hi strains. However, mutation at distinct sites within this area affected the interactions of distinct strains signifying the potential for tissue tropism via this receptor. Analyses of the molecular basis of interaction with human cell lines and purified CEA show that Hi strains, especially those belonging to Hi-aeg, interact with multiple CEACAMs. Because Neisseria meningitidis (Nm) strains are also known to bind at the CFG face of the receptor, we used Nm and Hi strains in co-infection experiments and demonstrate competition between these mucosal pathogens in colonization of target cells via CEACAMs.     

Occurrence of potentially invasive nontypeable Haemophilus influenzae strains isolating from children attending day-care centres and orphanages

Close contacts between children attending day-care centres, orphanages or similar institutions favours mutual transmission of infections with nontypeable H. influenzae (NTHi) strains. NTHi are transmitted via air-droplets or via direct contact with respiratory system exudates from nonsymptomatic carriers. The study aimed at monitoring of potentially invasive nontypeable H. influenzae strains of hmwA+ profile among children in day-care centres and orphanages. Monitoring of prevalence of strains of hmwA profile in a single day-care centres within 8 months confirmed high level of NTHi strains transmission including NTHi strains potentially invasive. It has been shown, that potentially invasive NTHi strains appear with different frequency in day-care centres and orphanages. It also points out that dissemination NTHi is easy in such an environment.     

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.     

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.     

Intranasal Immunization with Nontypeable Haemophilus influenzae Outer Membrane Vesicles Induces Cross-Protective Immunity in Mice

Haemophilus influenzae is a Gram-negative human-restricted bacterium that can act as a commensal and a pathogen of the respiratory tract. Especially nontypeable H. influenzae (NTHi) is a major threat to public health and is responsible for several infectious diseases in humans, such as pneumonia, sinusitis, and otitis media. Additionally, NTHi strains are highly associated with exacerbations in patients suffering from chronic obstructive pulmonary disease. Currently, there is no licensed vaccine against NTHi commercially available. Thus, this study investigated the utilization of outer membrane vesicles (OMVs) as a potential vaccine candidate against NTHi infections. We analyzed the immunogenic and protective properties of OMVs derived from various NTHi strains by means of nasopharyngeal immunization and colonization studies with BALB/c mice. The results presented herein demonstrate that an intranasal immunization with NTHi OMVs results in a robust and complex humoral and mucosal immune response. Immunoprecipitation revealed the most important immunogenic proteins, such as the heme utilization protein, protective surface antigen D15, heme binding protein A, and the outer membrane proteins P1, P2, P5 and P6. The induced immune response conferred not only protection against colonization with a homologous NTHi strain, which served as an OMV donor for the immunization mixtures, but also against a heterologous NTHi strain, whose OMVs were not part of the immunization mixtures. These findings indicate that OMVs derived from NTHi strains have a high potential to act as a vaccine against NTHi infections.     

Non-typeable Haemophilus influenzae adhere to and invade human bronchial epithelial cells via an interaction of lipooligosaccharide with the PAF receptor

Adherence and invasion are thought to be key events in the pathogenesis of non-typeable Haemophilus influenzae (NTHi). The role of NTHi lipooligosaccharide (LOS) in adherence was examined using an LOS-coated polystyrene bead adherence assay. Beads coated with NTHi 2019 LOS adhered significantly more to 16HBE14 human bronchial epithelial cells than beads coated with truncated LOS isolated from an NTHi 2019 pgmB:ermr mutant (P = 0.037). Adherence was inhibited by preincubation of cell monolayers with NTHi 2019 LOS (P = 0.0009), but not by preincubation with NTHi 2019 pgmB:ermr LOS. Competitive inhibition studies with a panel of compounds containing structures found within NTHi LOS suggested that a phosphorylcholine (ChoP) moiety was involved in adherence. Further experiments revealed that mutations affecting the oligosaccharide region of LOS or the incorporation of ChoP therein caused significant decreases in the adherence to and invasion of bronchial cells by NTHi 2019 (P < 0.01). Analysis of infected monolayers by confocal microscopy showed that ChoP+ NTHi bacilli co-localized with the PAF receptor. Pretreatment of bronchial cells with a PAF receptor antagonist inhibited invasion by NTHi 2109 and two other NTHi strains expressing ChoP+ LOS glycoforms exhibiting high reactivity with an anti-ChoP antibody on colony immunoblots. These data suggest that a particular subset of ChoP+ LOS glycoforms could mediate NTHi invasion of bronchial cells by means of interaction with the PAF receptor.     

Transmission of Haemophilus influenzae type b strains among children attending day-care centers and orphanages

Goal of the work was to evaluate the differences level among H. influenzae strains of b serotype isolated from children attending day-care centres and orphanages and among strains isolated from invasive infections. In the work PFGE in Hib strains transmission examination and for epidemiological studies among three sources of invasive infection was applied. Among 35 Hib strains tested and control strain 8 different pulsotyped were found. Among 21 strains colonising the nasopharynx of healthy children, and among 13 Hib strains isolated from cerebrospinal fluid, 6 and 1 pulsotypes were found, respectively. Results obtained show that healthy children might be colonizing with genotypes characteristic for Hib strains isolated from invasive infections. In this view wider Hib vaccination seems be expected, as Hib circulation is common.     

Study of genetic diversity of nontypeable Haemophilus influenzae strains isolated from healthy children and patients with infection symptoms

In the study the usefulness of genotyping methods for genetic variability examinations of non-typeable H. influenzae strains circulating in population as well as level the variability of NTHi strains isolated from healthy children and from symptomatic infection cases have been evaluated. Among genotyping methods evaluated, AFLP method of the MfeI/BglII set has been found most useful to study level of genetic variability of NTHi strains population. It has been shown that NTHi strains colonizing nasopharyngeal of healthy children present higher polymorphism level than strains isolated from patient with clinical symptoms of NTHi infection.     

A rapid and sensitive procedure for determination of 5-N-acetyl neuraminic acid in lipopolysaccharides of Haemophilus influenzae: a survey of 24 non-typeable H. influenzae strains

In view of the importance of 5-N-acetyl neuraminic acid in bacterial pathogenesis, a sensitive, reproducible and reliable method for the determination of 5-N-acetyl neuraminic acid levels in lipopolysaccharide (LPS) is described and applied to 24 different non-typeable Haemophilus influenzae (NTHi) strains. The method involves analysis by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) of terminal 5-N-acetyl neuraminic acid residues released by neuraminidase treatment of O-deacylated LPS. The procedure is relatively fast and the instrumental effort is moderate. The results of the procedure were compared with data obtained by 1H NMR and electrospray ionisation-mass spectrometry (ESI-MS). The analysis of LPS from 24 NTHi strains showed that 5-N-acetyl neuraminic acid was found to be a common constituent of LPS in NTHi. Only one strain (NTHi 432) did not show any sialylation. Molar ratios (LPS/5-N-acetyl neuraminic acid) ranged between 5/1 and 500/1. Several strains in which no 5-N-acetyl neuraminic acid could be determined by other methods including 1H NMR and ESI-MS were shown to contain 5-N-acetyl neuraminic acid by this HPAEC-PAD procedure. The method was applied to determine levels of terminal 5-N-acetyl neuraminic acid in LPS from NTHi strains grown under different conditions and mutant strains containing inactive LPS biosynthetic genes.     

Diagnostic difficulties in identification of Haemophilus influenzae colonising the nasopharynx of children

The frequency of capsulated or non-capsulated Haemophilus influenzae strains colonisation among children attending day-care centres or orphanages has been studied. Detection of capsulated or non-capsulated H. influenzae strains has been compared for agglutination test and PCR. Misdiagnosing of H. influenzae serotype with agglutination found in the study suggest that the frequency of Hib strains colonizing the nasopharynx might be lower that previously evaluated. Due to perspectives of the wider use of Hib Immunisation in the future, more efficient diagnosis scheme for identification/differentiation of capsulated and non-capsulated H. influenzae strain should be undertaken.     

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.     

Nontypeable Haemophilus influenzae: understanding virulence and commensal behavior

Haemophilus influenzae is genetically diverse and exists as a near-ubiquitous human commensal or as a pathogen. Invasive type b disease has been almost eliminated in developed countries; however, unencapsulated strains - nontypeable H. influenzae (NTHi) - remain important as causes of respiratory infections. Respiratory tract disease occurs when NTHi adhere to or invade respiratory epithelial cells, initiating one or more of several proinflammatory pathways. Biofilm formation explains many of the observations seen in chronic otitis media and chronic bronchitis. However, NTHi biofilms seem to lack a biofilm-specific polysaccharide in the extracellular matrix, a source of controversy regarding their relevance. Successful commensalism requires dampening of the inflammatory response and evasion of host defenses, accomplished in part through phase variation.     

A new structural type for Haemophilus influenzae lipopolysaccharide. Structural analysis of the lipopolysaccharide from nontypeable Haemophilus influenzae strain 486.

Structural elucidation of the sialylated lipopolysaccharide (LPS) of non-typeable Haemophilus influenzae (NTHi) strain 486 has been achieved by the application of high-field NMR techniques and ESI-MS along with composition and linkage analyses on O-deacylated LPS and oligosaccharide samples. It was found that the LPS contains the common element of H. influenzae, L-alpha-D-Hepp-(1-->2)-[PEtn-->6]-L-alpha-D-Hepp-(1-->3)-[beta-D-Glcp-(1-->4)]-L-alpha-D-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdop-(2-->6)-Lipid A, but instead of glycosyl substitution of the terminal heptose residue (HepIII) at the O2 position observed in other H. influenzae strains, HepIII is chain elongated at the O3 position by either lactose or sialyllactose (i.e. alpha-Neu5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp). The LPS is substituted by an O-acetyl group linked to the O2 position of HepIII and phosphocholine (PCho) which was located at the O6 position of a terminal alpha-D-Glcp residue attached to the central heptose, a molecular environment different from what has been reported earlier for PCho. In addition, minor substitution by O-linked glycine to the LPS was observed. By investigation of LPS from a lpsA mutant of NTHi strain 486, it was demonstrated that the lpsA gene product also is responsible for chain extension from HepIII in this strain. The involvement of lic1 in expression of PCho was established by investigation of a lic1 mutant of NTHi strain 486.     

Nontypable Haemophilus influenzae displays a prevalent surface structure molecular pattern in clinical isolates

Non-typable Haemophilus influenzae (NTHi) is a gram negative pathogen that causes acute respiratory infections and is associated with the progression of chronic respiratory diseases. Previous studies have established the existence of a remarkable genetic variability among NTHi strains. In this study we show that, in spite of a high level of genetic heterogeneity, NTHi clinical isolates display a prevalent molecular feature, which could confer fitness during infectious processes. A total of 111 non-isogenic NTHi strains from an identical number of patients, isolated in two distinct geographical locations in the same period of time, were used to analyse nine genes encoding bacterial surface molecules, and revealed the existence of one highly prevalent molecular pattern (lgtF+, lic2A+, lic1D+, lic3A+, lic3B+, siaA-, lic2C+, ompP5+, oapA+) displayed by 94.6% of isolates. Such a genetic profile was associated with a higher bacterial resistance to serum mediated killing and enhanced adherence to human respiratory epithelial cells.     

Multilocus sequence-typing for characterization of Moscow strains of Haemophilus influenzae type b

Haemophilius influenzae, type b (Hib) bacteria, were genotyped by multilocus sequence typing (MLST) using 5 loci (adk, fucK, mdh, pgi, recA). 42 Moscow Hib strains (including 38 isolates form cerebrospinal fluid of children, who had purulent meningitis in 1999-2001, and 4 strains isolated from healthy carriers of Hib), as well as 2 strains from Yekaterinburg were studied. In MLST a strain is characterized, by alleles and their combinations (an allele profile) referred to also as sequence-type (ST). 9 Sts were identified within the Russian Hib bacteria: ST-1 was found in 25 strains (57%), ST-12 was found in 8 strains (18%), ST-11 was found in 4 strains (9%) and ST-15 was found in 2 strains (4.5%); all other STs strains (13, 14, 16, 17, 51) were found in isolated cases (2.3%). A comparison of allelic profiles and of nucleotide sequences showed that 93% of Russian isolates, i.e. strain with ST-1, 11, 12, 13, 15 and 17, belong to one and the same clonal complex. 2 isolates from Norway and Sweden from among 7 foreign Hib strains studied up to now can be described as belonging to the same clonal complex; 5 Hib strains were different from the Russian ones.     

Nontypeable Haemophilus influenzae: Pathogenesis and Prevention

In this paper, we describe the ability of nontypeable Haemophilus influenzae (NTHi) to coexist with the human host and the devastating results associated with disruption of the delicate state of balanced pathogenesis, resulting in both acute and chronic respiratory tract infections. It has been seen that the strains of NTHi causing disease show a marked genetic and phenotypic diversity but that changes in the lipooligosaccharide (LOS) and protein size and antigenicity in chronically infected individuals indicate that individual strains of NTHi can remain and adapt themselves to avoid expulsion from their infective niche. The lack of reliance of NTHi on a single mechanism of attachment and its ability to interact with the host with rapid responses to its environment confirmed the success of this organism as both a colonizer and a pathogen. In vitro experiments on cell and organ cultures, combined with otitis media and pulmonary models in chinchillas, rats, and mice, have allowed investigations into individual interactions between NTHi and the mammalian host. The host-organism interaction appears to be a two-way process, with NTHi using cell surface structures to directly interact with the mammalian host and using secreted proteins and LOS to change the mammalian host in order to pave the way for colonization and invasion. Many experiments have also noted that immune system evasion through antigenic variation, secretion of enzymes and epithelial cell invasion allowed NTHi to survive for longer periods despite a specific immune response being mounted to infection. Several outer membrane proteins and LOS derivatives are discussed in relation to their efficacy in preventing pulmonary infections and otitis media in animals. General host responses with respect to age, genetic makeup, and vaccine delivery routes are considered, and a mucosal vaccine strategy is suggested.     

Interaction with C4b-binding protein contributes to nontypeable Haemophilus influenzae serum resistance

Complement evasion by various mechanisms is important for microbial virulence and survival in the host. One strategy used by some pathogenic bacteria is to bind the complement inhibitor of the classical pathway, C4b-binding protein (C4BP). In this study, we have identified a novel interaction between nontypeable Haemophilus influenzae (NTHi) and C4BP, whereas the majority of the typeable H. influenzae (a-f) tested showed no binding. One of the clinical isolates, NTHi 506, displayed a particularly high binding of C4BP and was used for detailed analysis of the interaction. Importantly, a low C4BP-binding isolate (NTHi 69) showed an increased deposition of C3b followed by reduced survival as compared with NTHi 506 when exposed to normal human serum. The main isoform of C4BP contains seven identical alpha-chains and one beta-chain linked together with disulfide bridges. Each alpha-chain is composed of eight complement control protein (CCP) modules and we have found that the NTHi 506 strain did not interact with rC4BP lacking CCP2 or CCP7 showing that these two CCPs are important for the binding. Importantly, C4BP bound to the surface of H. influenzae retained its cofactor activity as determined by analysis of C3b and C4b degradation. Taken together, NTHi interferes with the classical complement activation pathway by binding to C4BP.     

Binding of the non-typeable Haemophilus influenzae lipooligosaccharide to the PAF receptor initiates host cell signalling

Non-typeable Haemophilus influenzae (NTHi) invades host cells by binding of the platelet-activating factor (PAF) receptor via lipooligosaccharide (LOS) glycoforms containing phosphorylcholine (ChoP). The effect of NTHi infection on host cell signalling and its role in NTHi invasion was examined. The infection of human bronchial epithelial cells with NTHi 2019 increased cytosolic Ca²⁺ levels, and the invasion of bronchial cells by NTHi 2019 was inhibited by pretreatment with the cell-permeant intracellular Ca²⁺ chelator BAPTA-AM ( P  = 0.022) or thapsigargin ( P  = 0.016). Cytosolic inositol phosphate (IP) levels were also increased after infection with NTHi 2019 ( P  < 0.001), but not after infection with isogenic mutants expressing altered LOS glycoforms lacking ChoP. PAF receptor antagonist reduced NTHi 2019-stimulated IP production in a dose-dependent manner. NTHi 2019 invasion was inhibited by pertussis toxin (PTX) and the phosphatidylinositol-3-kinase inhibitors wortmannin and LY294002. The less invasive strain NTHi 7502 also initiated IP production, but was unaffected by PAF receptor antagonist or PTX. These data demonstrate that the binding of the PAF receptor by NTHi initiates receptor coupling to a PTX-sensitive heterotrimeric G protein complex, resulting in a multifactorial host cell signal cascade and bacterial invasion. Moreover, the data suggest that NTHi strains initiate cell signalling and invade by different mechanisms, and that invasion mediated by PAF receptor activation is more efficient than macropinocytosis.