Nontypeable Haemophilus Influenzae Strains Research Articles

Assessing the dual orientations of vaccine candidate P6 in nontypable Haemophilus influenzae (948.5)

Nontypable Haemophilus influenzae (NTHi) is a pathogenic bacterium that is known to cause otitis media and other respiratory diseases, mostly in children and older adults. P6, a protein vaccine candidate for protection against NTHi infection, has recently been shown to be inserted into the outer membrane of NTHi in two orientations; one being surface exposed and the other oriented facing in toward the peptidoglycan layer. For P6 to be considered a good vaccine candidate, it must be surface exposed and conserved in the majority of NTHi strains. Using confocal microscopy, flow cytometry, and a biotinylation technique, we have determined the common orientation of P6 in multiple strains of NTHi. Results suggest that a minority of the total P6 population is surface exposed. Implications for P6’s vaccine candidacy are discussed.Grant Funding Source: This study was supported by NIH NIDCD RO1 08671 (to MEP) and the Rochester Institute of Technology.

Antibodies to the HMW1/HMW2 and Hia Adhesins of Nontypeable Haemophilus influenzae Mediate Broad-Based Opsonophagocytic Killing of Homologous and Heterologous Strains

The HMW1/HMW2 and Hia proteins are highly immunogenic surface adhesins of nontypeable Haemophilus influenzae (NTHi). Approximately 75% of NTHi strains express HMW1/HMW2 adhesins, and most of the remaining 25% express an Hia adhesin. Our objective in this study was to assess the ability of antisera raised against purified HMW1/HMW2 proteins or recombinant Hia proteins to mediate opsonophagocytic killing of a large panel of unrelated NTHi strains. Native HMW1/HMW2 proteins were purified from three HMW1/HMW2-expressing NTHi strains. Recombinant fusion proteins expressing surface-exposed segments of either of two prototype Hia proteins were purified from Escherichia coli transformants. Immune sera raised in guinea pigs were assessed for their ability to mediate killing of NTHi in an opsonophagocytic assay with the HL-60 phagocytic cell line. The three HMW1/HMW2 antisera mediated killing of 22 of 65, 43 of 65, and 28 of 65 unrelated HMW1/HMW2-expressing NTHi strains, respectively. As a group, the three sera mediated killing of 48 of 65 HMW1/HMW2-expressing strains. The two Hia immune sera mediated killing of 12 of 24 and 13 of 24 unrelated Hia-expressing NTHi strains, respectively. Together, they mediated killing of 15 of 24 Hia-expressing strains. Neither the HMW1/HMW2 nor the Hia antisera mediated killing of NTHi expressing the alternative adhesin type. Antibodies directed against native HMW1/HMW2 proteins and recombinant Hia proteins are capable of mediating broad-based opsonophagocytic killing of homologous and heterologous NTHi strains. A vaccine formulated with a limited number of HMW1/HMW2 and Hia proteins might provide protection against disease caused by most NTHi strains.

Diversity of Nontypeable Haemophilus influenzae Strains Colonizing Australian Aboriginal and Non-Aboriginal Children

Nontypeable Haemophilus influenzae (NTHI) strains are responsible for respiratory-related infections which cause a significant burden of disease in Australian children. We previously identified a disparity in NTHI culture-defined carriage rates between Aboriginal and non-Aboriginal children (42% versus 11%). The aim of this study was to use molecular techniques to accurately determine the true NTHI carriage rates (excluding other culture-identical Haemophilus spp.) and assess whether the NTHI strain diversity correlates with the disparity in NTHI carriage rates. NTHI isolates were cultured from 595 nasopharyngeal aspirates collected longitudinally from asymptomatic Aboriginal (n=81) and non-Aboriginal (n=76) children aged 0 to 2 years living in the Kalgoorlie-Boulder region, Western Australia. NTHI-specific 16S rRNA gene PCR and PCR ribotyping were conducted on these isolates. Confirmation of NTHI by 16S rRNA gene PCR corrected the NTHI carriage rates from 42% to 36% in Aboriginal children and from 11% to 9% in non-Aboriginal children. A total of 75 different NTHI ribotypes were identified, with 51% unique to Aboriginal children and 13% unique to non-Aboriginal children (P<0.0001). The strain richness (proportion of different NTHI ribotypes) was similar for Aboriginal (19%, 65/346) and non-Aboriginal children (19%, 37/192) (P=0.909). Persistent carriage of the same ribotype was rare in the two groups, but colonization with multiple NTHI strains was more common in Aboriginal children than in non-Aboriginal children. True NTHI carriage was less than that estimated by culture. The Aboriginal children were more likely to carry unique and multiple NTHI strains, which may contribute to the chronicity of NTHI colonization and subsequent disease.

Molecular Epidemiology of Nontypeable Haemophilus influenzae Causing Community-Acquired Pneumonia in Adults

Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen which causes a variety of respiratory infections. The objectives of the study were to determine its antimicrobial susceptibility, to characterize the β-lactam resistance, and to establish a genetic characterization of NTHi isolates. Ninety-five NTHi isolates were analyzed by pulsed field gel electrophoresis (PFGE) and multi locus sequence typing (MLST). Antimicrobial susceptibility was determined by microdilution, and the ftsI gene (encoding penicillin-binding protein 3, PBP3) was PCR amplified and sequenced. Thirty (31.6%) isolates were non-susceptible to ampicillin (MIC≥2 mg/L), with 10 of them producing β-lactamase type TEM-1 as a resistance mechanism. After ftsI sequencing, 39 (41.1%) isolates showed amino acid substitutions in PBP3, with Asn526→ Lys being the most common (69.2%). Eighty-four patients were successfully treated with amoxicillin/clavulanic acid, ceftriaxone and levofloxacin. Eight patients died due either to aspiration or complication of their comorbidities. In conclusion, NTHi causing CAP in adults shows high genetic diversity and is associated with a high rate of reduced susceptibility to ampicillin due to alterations in PBP3. The analysis of treatment and outcomes demonstrated that NTHi strains with mutations in the ftsI gene could be successfully treated with ceftriaxone or fluoroquinolones.

The relationship between biofilm formations and capsule in Haemophilus influenzae

To evaluate the biofilm formation of non-typeable Haemophilus influenzae (NTHi) and H. influenzae type b (Hib) clinical isolates, we conducted the following study. Serotyping and polymerase chain reaction were performed to identify β-lactamase-negative ampicillin (ABPC)-susceptible (BLNAS), β-lactamase-negative ABPC-resistant (BLNAR), TEM-1 type β-lactamase-producing ABPC-resistant (BLPAR)-NTHi, and Hib. Biofilm formation was investigated by microtiter biofilm assay, as well as visually observation with a scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) in a continuous-flow chamber. As a result, totally 99 strains were investigated, and were classified into 4 groups which were 26 gBLNAS, 22 gBLNAR, 28 gBLPAR-NTHi and 23 Hib strains. The mean OD600 in the microtiter biofilm assay of gBLNAS, gBLNAR, gBLPAR-NTHi, and Hib strains were 0.57, 0.50, 0.34, and 0.08, respectively. NTHi strains were similar in terms of biofilm formations, which were observed by SEM and CLSM. Five Hib strains with the alternated type b cap loci showed significantly increased biofilm production than the other Hib strains. In conclusion, gBLNAS, gBLNAR, and gBLPAR-NTHi strains were more capable to produce biofilms compared to Hib strains. Our data suggested that resistant status may not be a key factor but capsule seemed to play an important role in H. influenzae biofilm formation.

Dual orientation of the outer membrane lipoprotein P6 of nontypeable haemophilus influenzae.

The majority of outer membrane (OM) lipoproteins in Gram-negative bacteria are tethered to the membrane via an attached lipid moiety and oriented facing in toward the periplasmic space; a few lipoproteins have been shown to be surface exposed. The outer membrane lipoprotein P6 from the Gram-negative pathogenic bacterium nontypeable Haemophilus influenzae (NTHi) is surface exposed and a leading vaccine candidate for prevention of NTHi infections. However, we recently found that P6 is not a transmembrane protein as previously thought (L. V. Michel, B. Kalmeta, M. McCreary, J. Snyder, P. Craig, M. E. Pichichero, Vaccine 29:1624-1627, 2011). Here we pursued studies to show that P6 has a dual orientation, existing infrequently as surface exposed and predominantly as internally oriented toward the periplasmic space. Flow cytometry using three monoclonal antibodies with specificity for P6 showed surface staining of whole NTHi cells. Confocal microscopy imaging confirmed that antibodies targeted surface-exposed P6 of intact NTHi cells and not internal P6 in membrane-compromised or dead cells. Western blots of two wild-type NTHi strains and a mutant NTHi strain that does not express P6 showed that P6 antibodies do not detect a promiscuous epitope on NTHi. Depletion of targets to nonlipidated P6 significantly decreased bactericidal activity of human serum. Protease digestion of surface-exposed P6 demonstrated that P6 is predominantly internally localized in a manner similar to its homologue Pal in Escherichia coli. We conclude that P6 of NTHi is likely inserted into the OM in two distinct orientations, with the predominant orientation facing in toward the periplasm.

A novel branching pattern in the lipopolysaccharide expressed by non-typeable Haemophilus influenzae strain 1232

We report the novel branching pattern in lipopolysaccharide (LPS) expressed by non-typeable Haemophilus influenzae (NTHi) strain 1232. The strain expressed the β-d-Glcp-(1→4)-[α-d-Galp-(1→4)-β-d-Galp-(1→7)]-d-α-d-Hepp-(1→6)-β-d-Glcp chain linked to the proximal heptose (HepI) of the conserved triheptosyl inner-core moiety of NTHi LPS: l-α-d-HepIIIp-(1→2)-[PEtn→6]-l-α-d-HepIIp-(1→3)-l-α-d-HepIp-(1→5)-[PPEtn→4]-α-Kdop-(2→6)-lipid A. The structure has been elucidated using NMR spectroscopy, electrospray ionization mass spectrometry (ESI-MS) and capillary electrophoresis coupled to electrospray ionization tandem mass spectrometry (CE-ESI-MSn) on O-deacylated LPS and core oligosaccharide (OS) materials, as well as HPLC-ESI-MSn on permethylated, dephosphorylated OS. It was also found that a tetrasaccharide unit bearing sialic acid [α-Neu5Ac-(2→3)-β-d-Galp-(1→4)-β-d-GlcNAcp-(1→3)-β-d-Galp-(1→] could substitute O-4 of the β-d-Glcp linked to HepI. In addition, the distal heptose (HepIII) was substituted by PCho→6-β-d-Galp-(1→ at the O-2 position.

Minimal biofilm eradication concentration of antimicrobial agents against nontypeable Haemophilus influenzae isolated from middle ear fluids of intractable acute otitis media

Nontypeable Haemophilus influenzae (NTHi) makes the clinical course of acute otitis media (AOM) intractable by forming a biofilm that may hamper the clearance of the bacteria from middle ear cavity. In this study, we evaluated the minimum biofilm eradication concentration (MBEC) of antimicrobial agents against biofilm-forming NTHi strains. Twelve NTHi strains isolated from middle ear fluids of Japanese children with intractable AOM before antimicrobial treatment were evaluated for MBEC of fluoroquinolones in comparison with those of β-lactams and macrolides. AMPC and CDTR required much higher concentration, i.e., high MBECs, to suppress the biofilm formation of NTHi. In contrast, fluoroquinolones followed by macrolides showed lower MBECs. MBEC would be a good parameter to infer the efficacies of antimicrobials against NTHi in biofilm.

Bactericidal antibody response against P6, protein D, and OMP26 of nontypeableHaemophilus influenzaeafter acute otitis media in otitis-prone children

The bactericidal antibody response to three nontypeable Haemophilus influenzae (NTHi) outer membrane proteins (D, P6, and OMP26) was studied in 24 otitis-prone children (aged 7-28months) after an acute otitis media (AOM) caused by NTHi. The study was carried out to understand the contribution of antigen-specific bactericidal antibody responses in the class of children who are most vulnerable to recurrent otitis media infections. Levels of protein D (P=0.005) and P6 (P=0.026) but not OMP26 antibodies were higher in bactericidal sera compared with nonbactericidal sera. For five (24%) and 16 (76%) of 21 bactericidal sera tested, removal of anti-protein D and P6 antibody, respectively, resulted in a two- to fourfold drop in bactericidal antibody. Antibodies to OMP26 did not make any contribution to the overall bactericidal activity in any serum samples. Eleven of 21 sera (52%) had bactericidal activity against a heterologous NTHi (86-028 NP) strain but the titers were significantly lower (P<0.05) as compared to the homologous strains. Future studies of protein D, P6, OMP26, and other potential NTHi vaccine antigens should include studies of bactericidal antibody in children who are otitis prone as a possible correlate of protection.

The role of Helicobacter pylori and histopathological findings in patients with dyspepsia

Background: Nontypeable Haemophilus influenzae (NTHi) is nown to be the causative agent of many chronic diseases such s otitis media, pneumonia, meningitis and sinusitis. Methods: In this work, a collection of 28 NTHi strains isoated from 12 different origin in Malaysia were examined on their ntimicrobial susceptibility profile to 10 antibiotics using the diskiffusion method. These strains were also analysed to determine or genotypic differences using a cost-effective typing method i.e. estriction fragment length polymorphisms (RFLP). In the RFLP nalysis, all of the isolates were amplified by using 16S primers nd digested with six restriction enzymes, namely EcoRI, HindIII, sp92I, BamHI, HaeIII and Hin1II. Results: Antimicrobial susceptibility test showed that 33% of he isolates were resistant to two or more of the tested antimirobials. NTHi isolates demonstrated resistance most frequently to mpicilin (43.75%), trimethoprim-sulfomethoxazole (31.25%) and .25% each to tetracycline, streptomycin and rifampicin. With the xception of Hin1II, the results of the digestion with the other five estriction enzymes were not reproducible and thus, the results ere excluded. Digestion with Hin1II produced between two to hree fragments in each isolate. The DNA fragments of 50–200bp ize could be clearly resolved into two RFLP patterns among the 28 solates. It was observed that the isolates fell into two clusters with enetic distances of >60% similarity. There were seven branches at -value of ≥0.9, representing at a genetic distance of more than 0% similarity of which these branches can be referred to as clonal roups. This study demonstrated that 27 of 28 (96.4%) NTHi strains hat were isolated from 12 distant locations had 100% similarity n their RFLP patterns. Few loci were detected and various other ombinations of restriction enzymes for typing of NTHi would be equired. Conclusion: This study showed the occurrence of antimicrobial esistance amongst NTHis isolated in Malaysia and although they ere geographical-distantly isolated, they are genetically related.

The structural diversity of lipopolysaccharide expressed by non-typeable Haemophilus influenzae strains 1158 and 1159

A heterogeneous population of glycoforms expressed by NTHi strains 1158 and 1159 has been elucidated using NMR spectroscopy and capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE-ESI-MS) on O-deacylated LPS (LPS-OH) and core oligosaccharide (OS) materials, as well as HPLC-ESI-MSn on dephosphorylated and methylated OS samples. The most abundant glycoform contained a disaccharide chain: PCho→7)-d-α-d-Hepp-(1→6)-β-d-Glcp linked to HepI from the common structural element of H. influenzae LPS: l-α-d-HepIIIp-(1→2)-[PEtn→6]-l-α-d-HepIIp-(1→3)-l-α-d-HepIp-(1→5)-[PPEtn→4]-α-Kdop-(2→6)-lipid A. Phosphocholine (PCho) was found at two positions in the LPS glycoforms; PCho substituted the 6-position of β-d-Glcp attached to HepIII and was also located at a novel position linked to d-α-d-Hepp; this latter position was determined by structural analysis of LPS from a 1158lpsA mutant strain. Additionally, HPLC-ESI-MSn experiments indicated glycoforms that have chain elongation from HepII, this was found only in glycoforms, which lack the additional heptose in the outer core region. Structural details of these glycoforms were confirmed by analyses of LPS from a 1158losB2 mutant strain; the losB2 gene is required for addition of the d,d-Hep to the outer core region in strain 1158.

Characterization of extended co-culture of non-typeable Haemophilus influenzae with primary human respiratory tissues

Non-typeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that comprise part of the normal flora of the human upper airway, but are also responsible for a number of mucosal infections such as otitis media and bronchitis. These infections often recur and can become chronic. To characterize the effect of long-term co-culture of NTHi with human tissues, we infected primary respiratory epithelial cells grown at the air–liquid interface with three NTHi strains over a range of 1–10 days. Scanning and transmission electron microscopy of tissues confirmed that intact NTHi were persisting paracellularly, while organisms observed in intracellular vacuoles appeared degraded. Furthermore, the apical surface and tight junctions of the infected tissues were undisturbed, with high transepithelial electrical resistances, while the basal cell layer displayed more junctional disorganization and wider intercellular spaces than the uninfected control tissues. Although the tissues elaborated the cytokine profile reported for NTHi-caused otitis media in vivo, there was little change in the dynamics of cytokine secretion over the time points tested. Finally, we report that NTHi strains released outer membrane vesicles (OMVs) during extended co-culture with the tissues, and show that these OMVs directly interact with host cell membranes.

Prevalence and Genetic Diversity of Nontypeable Haemophilus influenzae in the Respiratory Tract of Infants and Primary Caregivers

Nontypeable Haemophilus influenzae (NTHi) causes otitis media, sinusitis, and likely lower respiratory tract infections in children. Colonization, strain diversity, transmission, and antimicrobial susceptibility have implications for both children and their caregivers. For 13 months, we conducted a cross-sectional study of NTHi colonization. Upper respiratory tract cultures were performed in 273 infants and children 2 to 26 months of age and their primary caregivers. NTHi isolates were characterized by multilocus sequence typing (MLST), and antibiotic resistance was examined. Of the 273 infants, 44 (16.1%) were colonized with NTHi. Prevalence of NTHi varied from 14% in infants less than 6 months of age to 32% in infants between 19 and 26 months of age (P = 0.003). NTHi-colonized infants were more likely to attend day care (30% vs. 12%), have a recent respiratory infection (68% vs. 38%), have recently taken an antibiotic (27% vs. 9%), and have a primary caregiver who reported asthma (11% vs. 1%), compared with other infants (P < 0.01). In the 44 infants colonized with NTHi, we identified 33 different MLSTs. Of the 44 infant-primary caregiver dyads, 9 (20.5%) were colonized with NTHi, and 7 of these 9 shared identical NTHi strains. We also found beta-lactamase-negative NTHi with minimum inhibitory concentrations >2 μg/mL for amoxicillin and beta-lactamase-positive NTHi with minimum inhibitory concentrations >2 μg/mL for amoxicillin clavulanate. We found substantial diversity by MLST analysis among NTHi isolates from this community. Infant-primary caregiver dyads usually carried the same strain of NTHi, suggesting that infant-primary caregiver transmission is occurring.

Phylogenetic relatedness and diversity of non-typable Haemophilus influenzae in the nasopharynx and middle ear fluid of children with acute otitis media

The phylogenetic relationships of non-typable Haemophilus influenzae (NTHi) strains prospectively isolated from healthy children and children with acute otitis media (AOM) were analysed using multilocus sequence typing (MLST). A total of 165 NTHi isolates were collected over a 3.5 year time frame during 2006 through 2009. The strains were tested for β-lactamase production; 28.5% were positive. Seventy different NTHi sequence types (STs) were identified of which 29 (41.4%) were novel. NTHi strains did not show any phylogenetic grouping or clustering among asymptomatic colonizing strains or strains that caused AOM, or based on β-lactamase enzyme production. Evaluation of triplets and other siblings over time demonstrated relatively frequent genetic exchanges in NTHi isolates in vivo in a short time frame and subsequent transfer among children in a family. Comparison of the MLST STs isolated at different time points showed that in ~85% of the nasopharynx (NP) colonizations, NTHi strains cleared from the host within 3 months, that sequential colonization in the same child involved different strains in all cases except one, and that NP and middle ear isolates were identical STs in 84% of cases. In this first study of its type to our knowledge, we could not identify predominant MLST types among strains colonizing the NP versus those causing AOM or expressing a β-lactamase enzyme conferring penicillin resistance in children.

Sap Transporter Mediated Import and Subsequent Degradation of Antimicrobial Peptides in Haemophilus

Antimicrobial peptides (AMPs) contribute to host innate immune defense and are a critical component to control bacterial infection. Nontypeable Haemophilus influenzae (NTHI) is a commensal inhabitant of the human nasopharyngeal mucosa, yet is commonly associated with opportunistic infections of the upper and lower respiratory tracts. An important aspect of NTHI virulence is the ability to avert bactericidal effects of host-derived antimicrobial peptides (AMPs). The Sap (sensitivity to antimicrobial peptides) ABC transporter equips NTHI to resist AMPs, although the mechanism of this resistance has remained undefined. We previously determined that the periplasmic binding protein SapA bound AMPs and was required for NTHI virulence in vivo. We now demonstrate, by antibody-mediated neutralization of AMP in vivo, that SapA functions to directly counter AMP lethality during NTHI infection. We hypothesized that SapA would deliver AMPs to the Sap inner membrane complex for transport into the bacterial cytoplasm. We observed that AMPs localize to the bacterial cytoplasm of the parental NTHI strain and were susceptible to cytoplasmic peptidase activity. In striking contrast, AMPs accumulated in the periplasm of bacteria lacking a functional Sap permease complex. These data support a mechanism of Sap mediated import of AMPs, a novel strategy to reduce periplasmic and inner membrane accumulation of these host defense peptides.

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.

Effects of Bacterial Infection on Airway Antimicrobial Peptides and Proteins in COPD

Pathogenic bacteria colonize the airways of 30% to 40% of patients with COPD and cause approximately 50% of exacerbations. New strains of nontypeable Haemophilus influenzae (NTHI) and Moraxella catarrhalis are associated with exacerbations. Antimicrobial protein/peptides (AMPs) play important roles in innate lung defense against pathogens. To our knowledge, the changes in AMP baseline levels in respiratory secretions during bacterial colonization and exacerbation have not been described. The objective of this study was to elucidate the effects of the acquisition of a new strain of pathogenic bacteria on the airway levels of AMPs in patients with COPD. One hundred fifty-three samples from 11 patients were selected from COPD sputum samples collected prospectively over 6 years. Samples were grouped as culture-negative (no pathogenic bacteria), colonization, and exacerbation due to new strains of NTHI and M catarrhalis. Levels of lysozyme, lactoferrin, LL-37, and secretory leukocyte protease inhibitor (SLPI) were measured by enzyme-linked immunosorbent assay and compared among groups by paired analysis. Compared with baseline, sputum lysozyme levels were significantly lower during colonization and exacerbation by NTHI (P = .001 and P = .013, respectively) and M catarrhalis (P = .007 and P = .018, respectively); SLPI levels were lower with exacerbation due to NTHI and M catarrhalis (P = .002 and P = .004, respectively), and during colonization by M catarrhalis (P = 032). Lactoferrin levels did not change significantly; LL-37 levels were higher during exacerbation by NTHI and M catarrhalis (P = .001 and P = .018, respectively). Acquisition of NTHI and M catarrhalis is associated with significant changes in airway levels of AMPs, with larger changes in exacerbation. Airway AMP levels are likely to be important in pathogen clearance and clinical outcomes of infection in COPD.

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.

Expression of a new disialyllacto structure in the lipopolysaccharide of non-typeable Haemophilus influenzae

The structure of lipopolysaccharide (LPS) expressed by non-typeable Haemophilus influenzae (NTHi) strains 1008 and 1247 has been investigated by mass spectrometry and NMR analyses on O-deacylated LPS and core oligosaccharide material. Both strains express the conserved triheptosyl inner core, [l-α-d-Hepp-(1→2)-[PEtn→6]-l-α-d-Hepp-(1→3)-l-α-d-Hepp-(1→5)-[PPEtn→4]-α-Kdo-(2→6)-Lipid A] with PCho→6)-β-d-Glcp (GlcI) substituting the proximal heptose (HepI) at O-4. Strain 1247 expresses the common structural motifs of H. influenzae; globotetraose [β-d-GalpNAc-(1→3)-α-d-Galp-(1→4)-β-d-Galp-(1→4)-β-d-Glcp-(1→] and its truncated versions globoside [α-d-Galp-(1→4)-β-d-Galp-(1→4)-β-d-Glcp-(1→] and lactose [β-d-Galp-(1→4)-β-d-Glcp-(1→] linked to the terminal heptose of the inner core and GlcI. A genetically distinct NTHi strain, 1008, expresses identical structures to strain 1247 with the exception that it lacks GalNAc. A lpsA mutant of strain 1247 expressed LPS of reduced complexity that facilitated unambiguous structural determination of the oligosaccharide from HepI. By CE-ESI-MS/MS we identified disialylated glycoforms indicating disialyllactose [α-Neu5Ac-(2→8)-α-Neu5Ac-(2→3)-β-d-Gal-(1→4)-β-d-Glcp-(1→] as an extension from GlcI which is a novel finding for NTHi LPS.

Detailed structural features of lipopolysaccharide glycoforms in nontypeable Haemophilus influenzae strain 2019

We have investigated the structure of the lipopolysaccharide (LPS) of nontypeable Haemophilus influenzae (NT Hi) strain 2019, a prototype strain that is used for studies of NT Hi biology and disease. Analysis of LPS from wild type and lex2B, lpt3 and pgm mutant strains using NMR techniques and ESI-MS on O-deacylated LPS and core oligosaccharide material (OS), as well as ESI-MS n on permethylated dephosphorylated OS, confirmed the previously established structure in which lactose is linked to the proximal heptose (HepI) of the conserved triheptosyl inner-core moiety, l-α- d-Hep p-(1→2)-[ PEtn→6]- l-α- d-Hep p-(1→3)- l-α- d-Hep p-(1→5)-[ PPEtn→4]-α-Kdo-(2→6)-lipid A. Importantly, our data provide further structural detail whereby extensions from the middle heptose (HepII) are now characterized as β- d-Gal p-(1→4)-β- d-Glc p-(1→4)-α- d-Glc p-(1→3 and truncated versions thereof. PEtn substitutes O-3 of the distal heptose (HepIII) of the inner-core moiety. This PEtn substituent was absent in the lpt3 mutant indicating that Lpt3 is the transferase required to add PEtn to the distal heptose. Interestingly, in the lex2B mutant strain HepIII was found to be substituted at O-2 by β- d-Glc p which, in turn, can be further extended. Contrary to previous findings, LPS of the pgm mutant strain contained minor glycoforms having β- d-Glc p linked to O-4 of HepI and also glycoforms with an additional PEtn which could be assigned to HepIII. Acetate groups and one glycine residue further substitute HepIII in NT Hi 2019.