Adherence Of Nontypeable Haemophilus Influenzae Research Articles

Comparison of the adherence of nontypeable haemophilus influenzae to lung epithelial cells

ObjectiveNontypeable Haemophilus influenzae (NTHi) plays an important role in respiratory tract infections, and adherence to lung epithelial cells is the first step in lung infections. To explore the role of NTHi in childhood lung infections, a comparative study was conducted on the adherence of strains isolated from sputum culture and bronchoalveolar lavage fluid to A549 lung epithelial cells.MethodsHaemophilus influenzae strains were obtained from the sample bank of Shenzhen Children’s Hospital, and identified as NTHi via PCR detection of the capsule gene bexA. NTHi obtained from healthy children’s nasopharyngeal swabs culture were selected as the control group, and a comparative study was conducted on the adherence of strains isolated from sputum culture or bronchoalveolar lavage fluid of patients to A549 cells.ResultsThe adherence bacterial counts of NTHi isolated from the nasopharyngeal cultures of healthy children to A549 cells was 58.2 CFU. In patients with lung diseases, NTHi isolated from bronchoalveolar lavage fluid was 104.3 CFU, and from sputum cultures was 115.1 CFU, both of which were significantly higher in their adherence to A549 cells compared to the strains isolated from the healthy control group. There was no significant difference in adherence between the strains isolated from sputum cultures and bronchoalveolar lavage fluid (t = 0.5217, p = 0.6033).ConclusionNTHi played an important role in childhood pulmonary infections by enhancing its adherence to lung epithelial cells.

Adaptation of Nontypeable Haemophilus influenzae in Human Airways in COPD: Genome Rearrangements and Modulation of Expression of HMW1 and HMW2.

Chronic obstructive pulmonary disease (COPD) is a common debilitating disorder that is the third most common cause of death globally. Chronic lower airway infection by nontypeable Haemophilus influenzae (NTHi) in adults with COPD increases airway inflammation, causes increased symptoms, and accelerates progressive loss of lung function. Little is known about the mechanisms by which NTHi survives in COPD airways. To explore this question, the present study analyzes, in detail, 14 prospectively collected, serial isolates of a strain that persisted for 543 days in a patient with COPD, including analysis of four gap-free complete genomes. The NTHi genome underwent inversion of a ~400-kb segment three times during persistence. This inversion event resulted in switching of expression of the HMW1A and HMW2A adhesins as the inversion sites are in the promoter regions of HMW1 and HMW2. Regulation of the level of expression of HMW 1 and HMW2 in the human airways was controlled by the ~400-kb inversion and by 7-bp repeats in the HMW promoters. Analysis of knockout mutants of the persistent strain demonstrated that HMW1 and HMW2 proteins both function in the adherence of NTHi to human respiratory epithelial cells during persistence and that HMW1 also facilitates invasion of epithelial cells. An inverse relationship between biofilm formation and HMW1 expression was observed during persistence. This work advances understanding of the mechanisms of persistence of NTHi in COPD airways, which can inform the development of novel interventions to treat and prevent chronic NTHi infection in COPD. IMPORTANCE Nontypeable Haemophilus influenzae (NTHi) persists in the lower airways of adults with chronic obstructive pulmonary disease (COPD) for months to years, increasing airway inflammation that accelerates the progressive loss of lung function. Understanding the mechanisms of persistence in human airways by NTHi is critical in developing novel interventions. Here, in detail, we studied longitudinally collected sequential isolates of a strain of NTHi that persisted in an adult with COPD, including analysis of four gap-free genomes and knockout mutants to elucidate how the genome adapts in human airways. The NTHi genome underwent a genome rearrangement during persistence and this inversion impacted regulation of expression of key virulence phenotypes, including adherence to respiratory epithelial cells, invasion of epithelial cells and biofilm formation. These novel observations advance our understanding of the mechanisms of persistence of NTHi in the airways of adults with COPD.

Adherence of Nontypeable Haemophilus influenzae to Cells and Substrates of the Airway Is Differentially Regulated by Individual ModA Phasevarions.

Adherence of nontypeable Haemophilus influenzae (NTHi) to the host airway is an essential initial step for asymptomatic colonization of the nasopharynx, as well as development of disease. NTHi relies on strict regulation of multiple adhesins for adherence to host substrates encountered in the airway. NTHi encode a phase-variable cytoplasmic DNA methyltransferase, ModA, that regulates expression of multiple genes; a phasevarion (phase-variable regulon). Multiple modA alleles are present in NTHi, in which different alleles methylate a different DNA target, and each controls a different set of genes. However, the role of ModA phasevarions in regulating adherence of NTHi to the host airway is not well understood. This study therefore sought to investigate the role of four of the most prevalent ModA phasevarions in the regulation of adherence of NTHi to multiple substrates of the airway. Four clinical isolates of NTHi with unique modA alleles were tested in this study. The adherence of NTHi to mucus, middle ear epithelial cells, and vitronectin was regulated in a substrate-specific manner that was dependent on the ModA allele encoded. The adhesins Protein E and P4 were found to contribute to the ModA-regulated adherence of NTHi to distinct substrates. A better understanding of substrate-specific regulation of NTHi adherence by ModA phasevarions will allow identification of NTHi populations present at the site of disease within the airway and facilitate more directed development of vaccines and therapeutics. IMPORTANCE Nontypeable Haemophilus influenzae (NTHi) is a predominant pathogen of the human airway that causes respiratory infections such as otitis media (OM) and exacerbations in the lungs of patients suffering from chronic obstructive pulmonary disease (COPD). Due to the lack of a licensed vaccine against NTHi and the emergence of antibiotic-resistant strains, it is extremely challenging to target NTHi for treatment. NTHi adhesins are considered potential candidates for vaccines or other therapeutic approaches. The ModA phasevarions of NTHi play a role in the rapid adaptation of the pathogen to different environmental stress conditions. This study addressed the role of ModA phasevarions in the regulation of adherence of NTHi to specific host substrates found within the respiratory tract. The findings of this study improve our understanding of regulation of adherence of NTHi to the airway, which may further be used to enhance the potential of adhesins as vaccine antigens and therapeutic targets against NTHi.

Antimicrobial and Anti-Inflammatory Activity of Apple Polyphenol Phloretin on Respiratory Pathogens Associated With Chronic Obstructive Pulmonary Disease.

Bacterial infections contribute to accelerated progression and severity of chronic obstructive pulmonary disease (COPD). Apples have been associated with reduced symptoms of COPD and disease development due to their polyphenolic content. We examined if phloretin, an apple polyphenol, could inhibit bacterial growth and inflammation induced by the main pathogens associated with COPD. Phloretin displayed bacteriostatic and anti-biofilm activity against nontypeable Haemophilus influenzae (NTHi), Moraxella catarrhalis, Streptococcus pneumoniae, and to a lesser extent, Pseudomonas aeruginosa. In vitro, phloretin inhibited NTHi adherence to NCI-H292 cells, a respiratory epithelial cell line. Phloretin also exhibited anti-inflammatory activity in COPD pathogen-induced RAW 264.7 macrophages and human bronchial epithelial cells derived from normal and COPD diseased lungs. In mice, NTHi bacterial load and chemokine (C-X-C motif) ligand 1 (CXCL1), a neutrophil chemoattractant, was attenuated by a diet supplemented with phloretin. Our data suggests that phloretin is a promising antimicrobial and anti-inflammatory nutraceutical for reducing bacterial-induced injury in COPD.

Intercellular adhesion molecule 1 serves as a primary cognate receptor for the Type IV pilus of nontypeableHaemophilus influenzae

Nontypeable Haemophilus influenzae (NTHI) utilizes the Type IV pilus (Tfp) to adhere to respiratory tract epithelial cells thus colonizing its human host; however, the host cell receptor to which this adhesive protein binds is unknown. From a panel of receptors engaged by Tfp expressed by other bacterial species, we showed that the majority subunit of NTHI Tfp, PilA, bound to intercellular adhesion molecule 1 (ICAM1) and that this interaction was both specific and of high affinity. Further, Tfp-expressing NTHI inoculated on to polarized respiratory tract epithelial cells that expressed ICAM1 were significantly more adherent compared to Tfp-deficient NTHI or NTHI inoculated on to epithelial cells to which ICAM1 gene expression was silenced. Moreover, pre-incubation of epithelial cells with recombinant soluble PilA (rsPilA) blocked adherence of NTHI, an outcome that was abrogated by admixing rsPilA with ICAM1 prior to application on to the target cells. Epithelial cells infected with adenovirus or respiratory syncytial virus showed increased expression of ICAM1; this outcome supported augmented adherence of Tfp-expressing NTHI. Collectively, these data revealed the cognate receptor for NTHI Tfp as ICAM1 and promote continued development of a Tfp-targeted vaccine for NTHI-induced diseases of the airway wherein upper respiratory tract viruses play a key predisposing role.

Absence of high molecular weight proteins 1 and/or 2 is associated with decreased adherence among non-typeable Haemophilus influenzae clinical isolates

High molecular weight (Hmw) proteins 1 and 2, type IV pilin protein (PilA), outer-membrane protein P5 (OmpP5), Haemophilus protein D (Hpd) and Haemophilus adhesive protein (Hap) are surface proteins involved in the adherence of non-typeable Haemophilus influenzae. One hundred clinical isolates were evaluated for the presence of the genes encoding these proteins by PCR and for their adherence capacity (AC) to Detroit 562 nasopharyngeal cells (D562). The majority of isolates were from blood (77/100); other sites were also represented. Confluent D562 monolayers (1.2×10(5) cells per well) were inoculated with standardized minimal infective doses (m.o.i.) of 10(2), 10(3) or 10(4) c.f.u. per well. The AC was categorized as low (<10 %) or high (≥10 %) depending on the percentage of c.f.u. adhering per well. All the isolates evaluated showed adherence: 69/100 (69 %) demonstrated high adherence, while 31/100 (31 %) showed low adherence. Of all the genes evaluated, hmw1A and/or hmw2A were detected in 69/100 (69 %) of isolates. The presence of hmw1A and/or hmw2A was associated with increased adherence to D562 cells (P≤0.001). Dot immunoblots were performed to detect protein expression using mAbs 3D6, AD6 and 10C5. Among the high-adherence isolates (n = 69), 72 % reacted with 3D6 and 21 % with 10C5. Our data indicate that the absence of Hmw1 and/or Hmw2 was associated with decreased adherence to D562 cells.

Abrogation of nontypeable Haemophilus influenzae Protein D function reduces phosphorylcholine decoration, adherence to airway epithelial cells, and fitness in a chinchilla model of otitis media

The pneumococcal polysaccharide conjugate vaccine which includes a nonacylated protein D carrier from Haemophilus influenzae has been recently licensed for use in many countries. While this vaccine is protective against nontypeable Haemophilus influenzae (NTHI)-induced acute otitis media (OM), the mechanism underlying this protective efficacy is not yet fully understood. Protein D/glycerophosphodiester phosphodiesterase (PD/GlpQ) is an outer membrane lipoprotein expressed by NTHI that has been ascribed several functions, including host cell adherence and phosphorylcholine (PCho) acquisition. We found that a pd/glpQ NTHI mutant exhibited reduced adherence to airway epithelial cells, diminished phosphorylcholine (PCho) decoration of biofilms, and compromised fitness during experimental acute OM compared to the parent strain. We also found that exposure of NTHI to antibodies directed against the vaccine formulation recapitulated the PCho decoration and NTHI adherence phenotypes exhibited by PD/GlpQ-deficient NTHI, providing at least two likely mechanisms by which the pneumococcal polysaccharide-PD/GlpQ conjugate vaccine induces protection from NTHI-induced OM.

The Effects of Disodium Cromoglycate on Enhanced Adherence of Haemophilus influenzae to A549 Cells Infected With Respiratory Syncytial Virus

Nontypeable Haemophilus influenzae (NTHi) secondary infection often complicates respiratory syncytial virus (RSV) infections. Previous studies have revealed that RSV infections enhance NTHi adherence to airway epithelial cells. In this study, we investigated the effects of disodium cromoglycate (DSCG) and corticosteroids, which are frequently used for the treatment of wheezing often related to RSV infections, on the adherence of NTHi to RSV-infected A549 cells. DSCG inhibited enhanced adherence of NTHi to RSV-infected A549 cells, whereas dexamethasone (Dex) and fluticasone propionate (Fp) did not. DSCG suppressed the expression of ICAM-1, which is one of the NTHi receptors. Furthermore, DSCG exhibited an inhibitory effect on RSV infections. It is suggested that DSCG exerts an anti-RSV effect, and consequently attenuates the expression of NTHi receptors.

Nontypeable Haemophilus influenzae isolated from intractable acute otitis media internalized into cultured human epithelial cells

Objectives The aim of this study is to examine the internalization of nontypeable Haemophilus influenzae (NTHi) into human epithelial cells. Methods Bactericidal assay was applied to examine the effects of antibiotics against cell-adherent NTHi using HEp-2 cells. A trans-well chamber assay was applied to examine the internalization and penetration of NTHi using Detroit562 cells. Results The adherence of NTHi to HEp-2 cells was noted after 2 h of incubation. Azithromycin had a strong bactericidal effect against both cell-associated and non-adherent NTHi, while ceftriaxone did not show bactericidal effects on NTHi adhered to the HEp-2 cells. Three (60.0%) out of five NTHi isolates from the nasopharynx of children with intractable acute otitis media (AOM) internalized into and subsequently penetrated through the epithelial cells at various degrees. Azithromycin had a strong bactericidal effect against the cell-internalized NTHi, while ceftriaxone was bactericidal only against extracellular NTHi. Conclusion The potential of NTHi as the intracellular pathogen may contribute to the persistent existence of this pathogen that result in the prolonged and intractable clinical course of AOM. Azithromycin may be a therapeutically significant antibiotic for patients with prolonged respiratory tract infections due to NTHi.

NontypeableHaemophilus influenzaeAdhesin Protein E: Characterization and Biological Activity

The adhesin protein E (PE) of the human respiratory pathogen nontypeable Haemophilus influenzae (NTHi) exists in all clinical isolates. In the present study, NTHi adherence to epithelial cells of various origins was further analyzed. The number of intraepithelial PE-deficient NTHi was decreased compared with PE-expressing NTHi. Interestingly, PE-expressing NTHi or Escherichia coli transformants, in addition to soluble recombinant PE22-160 without a lipid moiety, induced a proinflammatory cell response. The adhesive PE domain was defined within PE84-108, and preincubation of epithelial cells with this peptide blocked adhesion of several clinical NTHi isolates. Mice immunized with PE84-108 cleared NTHi up to 8-fold more efficiently on pulmonary challenge than did mice immunized with a control peptide. Finally, anti-PE mouse antibodies from vaccinated mice prevented NTHi adhesion. Our data suggest that the ubiquitous adhesin PE plays an important role in the pathogenesis of NTHi infection.

Nontypeable Haemophilus influenzae and Streptococcus pneumoniae bind respiratory syncytial virus glycoprotein

Respiratory syncytial virus (RSV) infection is associated with secondary bacterial infections caused by nontypeable Haemophilus influenzae (NTHi) and Streptococcus pneumoniae. The pathogenesis of these complications is not completely understood; however, viral infection of respiratory epithelial cells promotes colonization by these bacteria. In the present study, RSV virions associated with NTHi and pneumococci in an inoculum-dependent manner in a fluid-phase binding assay. Adherence of NTHi and S. pneumoniae to epithelial cells transiently expressing RSV G glycoprotein was 2- and 2.2-fold higher, respectively, than adhesion to cells transfected with the vector alone (P <0.01). Furthermore, 4.6- and 6.2-fold larger numbers of NTHi and pneumococci bound to cells expressing a membrane-bound full-length RSV G protein than to cells expressing a truncated non-membrane-bound protein (P <or=0.005). Pre-incubating cells expressing membrane-bound G protein with blocking anti-RSV G antibodies reduced bacterial adherence by 78-84 % (P <or=0.005). These studies demonstrate that RSV G protein is a receptor for both NTHi and S. pneumoniae. Strategies to prevent this interaction may reduce the incidence of secondary bacterial complications of RSV infection.

Biological activities of antibodies elicited by lipooligosaccharide based-conjugate vaccines of nontypeable Haemophilus influenzae in an otitis media model

Vaccination of chinchillas with nontypeable Haemophilus influenzae (NTHi) lipooligosaccharide (LOS) conjugates protected against otitis media. Correlations between the levels of conjugate-induced LOS antibodies (Abs) in sera and middle ear fluids (MEFs) and Ab-mediated biological functions and protection were examined. Following parenteral vaccination and middle ear challenge, all vaccinated animals, but none of the controls, had high titers of anti-LOS in their sera and MEFs. There was a correlation between the levels of anti-LOS IgG+M, IgG or IgA in the sera and in the MEFs ( P<0.001). An inverse correlation was found between the level of serum IgG+M and bacterial counts and between the levels of MEF Abs and bacterial counts at the early postchallenge stage ( P<0.05). Of the 39 vaccinated animals, 44% showed complete protection against otitis media, 46% (18/39) of their sera inhibited adherence of NTHi to human epithelial cells, 49% (19/39) demonstrated bactericidal activity and 49% (19/39) showed opsonophagocytic activity. In contrast, none of the controls (19) were protected, none of their sera inhibited bacterial adherence or had bactericidal activity and only 21% showed opsonophagocytosis. Our interpretation is that vaccine-induced LOS Abs transuded into the middle ear and conferred immunity to NTHi by binding to LOS of NTHi, inhibition of NTHi adherence to epithelial cells and complement-mediated bacteriolysis (or opsonophagocytosis).

Specific mucosal immunity and enhanced nasopharyngeal clearance of nontypeable Haemophilus influenzae after intranasal immunization with outer membrane protein P6 and cholera toxin

Nontypeable Haemophilus influenzae (NTHi) is one of the leading pathogens in otitis media. Studies of vaccines against NTHi have focused on outer membrane proteins (OMPs). One outer membrane protein P6 is highly conserved among strains and is an attractive candidate for a subunit bacterial vaccine. In this study, mucosal immunity induced by intranasal immunization with P6 and cholera toxin (CT) was investigated in a mouse model. Intranasal immunization with P6 and CT evoked a good mucosal IgA as well as a systemic IgG response against P6. On the other hand, intranasal immunization with P6 alone induced a weak mucosal IgA response. Enzyme linked immunospot assay detected anti-P6 specific antibody producing cells in the nasopharyngeal mucosa of immunized mice. The protective response of intranasal immunization was demonstrated by enhancement of nasopharyngeal clearance of NTHi and inhibition of adherence of NTHi to cultured human epithelial cells. Based on these results, intranasal immunization with P6 and CT may be an effective approach to protect human from H. influenzae infections in the upper respiratory tract.

Adherence of non-typeableHaemophilus influenzaepromotes reorganization of the actin cytoskeleton in human or chinchilla epithelial cellsin vitro

Non-typeableHaemophilus influenzae(NTHi) are opportunistic mucosal pathogens which adhere to epithelial cells via a variety of non-specific and specific interactions. Several adhesins have been identified and while the complimentary receptor(s) for each of these adhesins has not yet been fully characterized, it is widely accepted that adherence is an absolute prerequisite for disease. Several reports have indicated that NTHi can also be internalized and reside intracellularly. For this to occur, NTHi must be taken up by mucosal epithelial cells lining the respiratory tract. We have noted, by TEM, that adherent NTHi overlie an electron dense area in the cell membrane of human epithelial cells which is associated with a localized complex assembly of cytoskeletal fibers in the eukaryotic cytoplasm. We thus examined the potential involvement of cytoskeletal actin in this phenomenon via FITC-phalloidin labeling of respiratory tract epithelial cells which had been incubated with several clinical isolates of NTHi. Strong punctate fluorescence was coincident with adherent NTHi to both human oropharyngeal and chinchilla middle ear epithelial cells. This reactivity was similar to the discrete fluorescent spots observed with enteropathogenicEscherichia coliwhich were adhered to HeLa cells. In contrast, none of the NTHi isolates tested induced actin polymerization in cells of endothelial origin. While the exact mechanisms involved are yet to be elucidated, our data indicated that actin nucleation was coincident with NTHi adherence.

Adherence of Nontypeable Haemophilus Influenzae to Respiratory Epithelium of Otitis-Prone and Normal Children

Three hundred six children were enrolled at birth in a prospective study of otitis media and followed up for 2 years. Adherence of nontypeable Haemophilus influenzae to buccal epithelial cells was compared between otitis-prone children and age- and sex-matched normal controls at birth, 1 year, and 2 years. The mean +/- SD/median percent adherence was similar for the two groups at birth (1.6 +/- 2.3/1.0 versus 1.2 +/- 1.4/1.0; NS) and at 2 years (1.6 +/- 1.7/1.5 versus 2.1 +/- 2.1/1.5; NS). At 1 year of age the adherence rate for the otitis-prone group (2.4 +/- 2.6/1.0) was statistically greater than that for the control group (1.0 +/- 1.3/0.0; p < .02). Because this difference is probably clinically insignificant, other explanations must be sought for the increased colonization rates of nontypeable H influenzae observed in otitis-prone children.

Functional and ultrastructural effects of nontypeable Haemophilus influenzae in a hamster trachea organ culture system.

A hamster trachea organ culture system was utilized to evaluate quantitatively the effects of a strain of nontypeable Haemophilus influenzae (NTHI) and culture supernatants of the same strain on ciliary activity. Tracheal explants were maintained in organ culture for 96 to 144 h and ciliary activity was observed daily with an inverted microscope. Explants continuously exposed to a strain of NTHI had a progressive decline in ciliary activity which was significantly lower than uninfected controls evaluated concomitantly by 48 h of exposure and thereafter. Histologic studies revealed a progressive degeneration of mucosal cells and exfoliation of ciliated cells. Scanning electron microscopy showed little adherence of NTHI to the mucosal surface. Sterile broth cultures of NTHI and supernatants of organ cultures infected with the same NTHI strain had no adverse effect on ciliary activity. Infected tracheal explants treated with ampicillin 24, 48, or 72 h after continuous bacterial challenge had no significant decline in ciliary activity compared to controls. The lack of adherence and the histologic changes observed when hamster trachea cultures were infected with NTHI suggested a toxin might mediate the damage observed. Broth and organ culture supernatants, however, produced no damage. Therefore, further studies are needed to determine the role, if any, of a toxin in the production of damage to hamster tracheal explants by NTHI.