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1
Jiang, Zili, Nobuo Nagata, Edgar Molina, Lauren O. Bakaletz, Hal Hawkins, and Janak A. Patel. "Fimbria-Mediated Enhanced Attachment of NontypeableHaemophilus influenzae to Respiratory Syncytial Virus-Infected Respiratory Epithelial Cells." Infection and Immunity 67, no. 1 (January 1, 1999): 187–92. http://dx.doi.org/10.1128/iai.67.1.187-192.1999.
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ABSTRACT Respiratory syncytial virus (RSV) infection is known to predispose children to otitis media and sinusitis due to bacteria such as nontypeable Haemophilus influenzae (NTHI). In this study, we investigated the role of NTHI surface outer membrane protein P5-homologous fimbriae (P5-fimbriae) in attachment to RSV-exposed A549 epithelial cells. Analysis by fluorescence flow cytometry showed that a live P5-fimbriated NTHI strain (NTHIF+) attached to a higher proportion of RSV-exposed A549 cells than to control cells (mean, 68% for RSV versus 29% for control; P = 0.008), while attachment of the P5-fimbriae-deficient isogenic mutant strain (NTHIF−) was significantly lower than in control cells and rose only slightly following RSV exposure (mean, 17% for RSV versus 10% for control, P = 0.229). Attachment of NTHIF+ did not correlate with the amount of RSV antigen expressed by A549 cells. Furthermore, paraformaldehyde-fixed NTHIF+ also demonstrated an enhanced binding to RSV-exposed cells. Observations by transmission electronic microscopy showed that the mean number of bacteria attached per 100 RSV-exposed A549 cells was higher for NTHIF+ than NTHIF− (99 versus 18; P < 0.001). No intracellular bacteria were identified. UV-irradiated conditioned supernatants collected from RSV-infected A549 cultures (UV-cRSV) also enhanced the attachment of NTHIF+ to A549, suggesting the presence of a preformed soluble mediator(s) in UV-cRSV that enhances the expression of receptors for P5-fimbriae on A549 cells. In summary, RSV infection significantly enhances NTHI attachment to respiratory epithelial cells. P5-fimbria is the critical appendage of NTHI that participates in this attachment. In clinical settings, blocking of the P5-fimbria-mediated attachment of NTHIF+ by passive or active immunity may reduce the morbidity due to NTHI during RSV infection.
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Satola, Sarah W., Brooke Napier, and Monica M. Farley. "Association of IS1016 with the hia Adhesin Gene and Biotypes V and I in Invasive Nontypeable Haemophilus influenzae." Infection and Immunity 76, no. 11 (September 15, 2008): 5221–27. http://dx.doi.org/10.1128/iai.00672-08.
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ABSTRACT A subset of invasive nontypeable Haemophilus influenzae (NTHI) strains has evidence of IS1016, an insertion element associated with division I H. influenzae capsule serotypes. We examined IS1016-positive invasive NTHI isolates collected as part of Active Bacterial Core Surveillance within the Georgia Emerging Infections Program for the presence or absence of hmw1 and hmw2 (two related adhesin genes that are common in NTHI but absent in encapsulated H. influenzae) and hia (homologue of hsf, an encapsulated H. influenzae adhesin gene). Isolates were serotyped using slide agglutination, confirmed as NTHI strains using PCR capsule typing, and biotyped. Two hundred twenty-nine invasive NTHI isolates collected between August 1998 and December 2006 were screened for IS1016; 22/229 (9.6%) were positive. Nineteen of 201 previously identified IS1016-positive invasive NTHI isolates collected between January 1989 and July 1998 were also examined. Forty-one IS1016-positive and 56 randomly selected IS1016-negative invasive NTHI strains were examined. The hia adhesin was present in 39 of 41 (95%) IS1016-positive NTHI strains and 1 of 56 (1.8%) IS1016-negative NTHI strains tested; hmw (hmw1, hmw2, or both) was present in 50 of 56 (89%) IS1016-negative NTHI isolates but in only 5 of 41 (12%; all hmw2) IS1016-positive NTHI isolates. IS1016-positive NTHI strains were more often biotype V (P < 0.001) or biotype I (P = 0.04) than IS1016-negative NTHI strains, which were most often biotype II. Pulsed-field gel electrophoresis revealed the expected genetic diversity of NTHI with some clustering based on IS1016, hmw or hia, and biotypes. A significant association of IS1016 with biotypes V and I and the presence of hia adhesins was found among invasive NTHI. IS1016-positive NTHI strains may represent a unique subset of NTHI strains, with characteristics more closely resembling those of encapsulated H. influenzae.
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Euba, Begoña, Javier Moleres, Cristina Viadas, Montserrat Barberán, Lucía Caballero, María-Jesús Grilló, José Antonio Bengoechea, et al. "Relationship between Azithromycin Susceptibility and Administration Efficacy for Nontypeable Haemophilus influenzae Respiratory Infection." Antimicrobial Agents and Chemotherapy 59, no. 5 (February 23, 2015): 2700–2712. http://dx.doi.org/10.1128/aac.04447-14.
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ABSTRACTNontypeableHaemophilus influenzae(NTHI) is an opportunistic pathogen that is an important cause of acute exacerbations of chronic obstructive pulmonary disease (AECOPD). COPD is an inflammatory disease of the airways, and exacerbations are acute inflammatory events superimposed on this background of chronic inflammation. Azithromycin (AZM) is a macrolide antibiotic with antibacterial and anti-inflammatory properties and a clinically proven potential for AECOPD prevention and management. Relationships between AZM efficacy and resistance by NTHI and between bactericidal and immunomodulatory effects on NTHI respiratory infection have not been addressed. In this study, we employed two pathogenic NTHI strains with different AZM susceptibilities (NTHI 375 [AZM susceptible] and NTHI 353 [AZM resistant]) to evaluate the prophylactic and therapeutic effects of AZM on the NTHI-host interplay. At the cellular level, AZM was bactericidal toward intracellular NTHI inside alveolar and bronchial epithelia and alveolar macrophages, and it enhanced NTHI phagocytosis by the latter cell type. These effects correlated with the strain MIC of AZM and the antibiotic dose. Additionally, the effect of AZM on NTHI infection was assessed in a mouse model of pulmonary infection. AZM showed both preventive and therapeutic efficacies by lowering NTHI 375 bacterial counts in lungs and bronchoalveolar lavage fluid (BALF) and by reducing histopathological inflammatory lesions in the upper and lower airways of mice. Conversely, AZM did not reduce bacterial loads in animals infected with NTHI 353, in which case a milder anti-inflammatory effect was also observed. Together, the results of this work link the bactericidal and anti-inflammatory effects of AZM and frame the efficacy of this antibiotic against NTHI respiratory infection.
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Juneau, Richard A., Bing Pang, Kristin E. D. Weimer, Chelsie E. Armbruster, and W. Edward Swords. "NontypeableHaemophilus influenzaeInitiates Formation of Neutrophil Extracellular Traps." Infection and Immunity 79, no. 1 (October 18, 2010): 431–38. http://dx.doi.org/10.1128/iai.00660-10.
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ABSTRACTNontypeableHaemophilus influenzae(NTHI) is a leading cause of otitis media infections, which are often chronic and/or recurrent in nature. NTHI and other bacterial species persistin vivowithin biofilms during otitis media and other persistent infections. These biofilms have a significant host component that includes neutrophil extracellular traps (NETs). These NETs do not mediate clearance of NTHI, which survives within NET structures by means of specific subpopulations of lipooligosaccharides on the bacterial surface that are determinants of biofilm formationin vitro. In this study, the ability of NTHI and NTHI components to initiate NET formation was examined using anin vitromodel system. Both viable and nonviable NTHI strains were shown to promote NET formation, as did preparations of bacterial DNA, outer membrane proteins, and lipooligosaccharide (endotoxin). However, only endotoxin from a parental strain of NTHI exhibited equivalent potency in NET formation to that of NTHI. Additional studies showed that NTHI entrapped within NET structures is resistant to both extracellular killing within NETs and phagocytic killing by incoming neutrophils, due to oligosaccharide moieties within the lipooligosaccharides. Thus, we concluded that NTHI elicits NET formation by means of multiple pathogen-associated molecular patterns (most notably endotoxin) and is highly resistant to killing within NET structures. These data support the conclusion that, for NTHI, formation of NET structures may be a persistence determinant by providing a niche within the middle-ear chamber.
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Avadhanula, Vasanthi, Carina A. Rodriguez, Glen C. Ulett, Lauren O. Bakaletz, and Elisabeth E. Adderson. "Nontypeable Haemophilus influenzae Adheres to Intercellular Adhesion Molecule 1 (ICAM-1) on Respiratory Epithelial Cells and Upregulates ICAM-1 Expression." Infection and Immunity 74, no. 2 (February 2006): 830–38. http://dx.doi.org/10.1128/iai.74.2.830-838.2006.
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ABSTRACT Nontypeable Haemophilus influenzae (NTHI) is an important respiratory pathogen. NTHI initiates infection by adhering to the airway epithelium. Here, we report that NTHI interacts with intracellular adhesion molecule 1 (ICAM-1) expressed by respiratory epithelial cells. A fourfold-higher number of NTHI bacteria adhered to Chinese hamster ovary (CHO) cells transfected with human ICAM-1 (CHO-ICAM-1) than to control CHO cells (P ≤ 0.005). Blocking cell surface ICAM-1 with specific antibody reduced the adhesion of NTHI to A549 respiratory epithelial cells by 37% (P = 0.001) and to CHO-ICAM-1 cells by 69% (P = 0.005). Preincubating the bacteria with recombinant ICAM-1 reduced adhesion by 69% (P = 0.003). The adherence to CHO-ICAM-1 cells of NTHI strains deficient in the adhesins P5, P2, HMW1/2, and Hap or expressing a truncated lipooligosaccharide was compared to that of parental strains. Only strain 1128f−, which lacks the outer membrane protein (OMP) P5-homologous adhesin (P5 fimbriae), adhered less well than its parental strain. The numbers of NTHI cells adhering to CHO-ICAM-1 cells were reduced by 67% (P = 0.009) following preincubation with anti-P5 antisera. Furthermore, recombinant ICAM bound to an OMP preparation from strain 1128f+, which expresses P5, but not to that from its P5-deficient mutant, confirming a specific interaction between ICAM-1 and P5 fimbriae. Incubation of respiratory epithelial cells with NTHI increased ICAM-1 expression fourfold (P = 0.001). Adhesion of NTHI to the respiratory epithelium, therefore, upregulates the expression of its own receptor. Blocking interactions between NTHI P5 fimbriae and ICAM-1 may reduce respiratory colonization by NTHI and limit the frequency and severity of NTHI infection.
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Atto, Brianna, Roger Latham, Dale Kunde, David A. Gell, and Stephen Tristram. "In Vitro Anti-NTHi Activity of Haemophilin-Producing Strains of Haemophilus haemolyticus." Pathogens 9, no. 4 (March 25, 2020): 243. http://dx.doi.org/10.3390/pathogens9040243.
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Nontypeable Haemophilus influenzae (NTHi) is a leading causative organism of opportunistic respiratory tract infections. However, there are currently no effective vaccination strategies, and existing treatments are compromised by antibiotic resistance. We previously characterized Haemophilus haemolyticus (Hh) strains capable of producing haemophilin (HPL), a heme-binding protein that restricts NTHi growth by limiting its access to an essential growth factor, heme. Thus, these strains may have utility as a probiotic therapy against NTHi infection by limiting colonization, migration and subsequent infection in susceptible individuals. Here, we assess the preliminary feasibility of this approach by direct in vitro competition assays between NTHi and Hh strains with varying capacity to produce HPL. Subsequent changes in NTHi growth rate and fitness, in conjunction with HPL expression analysis, were employed to assess the NTHi-inhibitory capacity of Hh strains. HPL-producing strains of Hh not only outcompeted NTHi during short-term and extended co-culture, but also demonstrated a growth advantage compared with Hh strains unable to produce the protein. Additionally, HPL expression levels during competition correlated with the NTHi-inhibitory phenotype. HPL-producing strains of Hh demonstrate significant probiotic potential against NTHi colonization in the upper respiratory tract, however, further investigations are warranted to demonstrate a range of other characteristics that would support the eventual development of a probiotic.
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Lewnard, Joseph A., Noga Givon-Lavi, and Ron Dagan. "Interaction With Nontypeable Haemophilus influenzae Alters Progression of Streptococcus pneumoniae From Colonization to Disease in a Site-Specific Manner." Journal of Infectious Diseases 220, no. 8 (June 23, 2019): 1367–76. http://dx.doi.org/10.1093/infdis/jiz312.
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Abstract Background Pneumococci and nontypeable Haemophilus influenzae (NTHi) often cocolonize children. The impact of species interactions on disease risk across the upper respiratory mucosa is not known. Methods We analyzed data from 4104 acute conjunctivitis (AC) cases, 11 767 otitis media (OM) cases, and 1587 nasopharyngeal specimens collected from Israeli children before pneumococcal conjugate vaccine introduction. We compared pneumococcal serotype distributions with NTHi present and absent, and compared single-species and mixed-species rates of serotype-specific progression from colonization to AC and OM. Results Pneumococcal serotypes causing single-species OM (NTHi absent) were less diverse than colonizing serotypes and also less diverse than those causing mixed-species OM; colonizing and OM-causing pneumococcal serotype distributions were more similar to each other with NTHi present than with NTHi absent. In contrast, serotype diversity did not differ appreciably between colonizing and AC-causing pneumococci, regardless of NTHi co-occurrence. The similarity of colonizing and AC-causing pneumococcal serotype distributions was consistent in the presence and absence of NTHi. Differences in rates that pneumococcal serotypes progressed from colonization to disease were reduced in both AC and OM when NTHi was present. Conclusions Interactions with NTHi may alter progression of pneumococcal serotypes to diseases of the upper respiratory mucosa in a site-specific manner.
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Morey, Pau, Victoria Cano, Pau Martí-Lliteras, Antonio López-Gómez, Verónica Regueiro, Carles Saus, José Antonio Bengoechea, and Junkal Garmendia. "Evidence for a non-replicative intracellular stage of nontypable Haemophilus influenzae in epithelial cells." Microbiology 157, no. 1 (January 1, 2011): 234–50. http://dx.doi.org/10.1099/mic.0.040451-0.
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Nontypable Haemophilus influenzae (NTHi) is a Gram-negative, non-capsulated human bacterial pathogen, a major cause of a repertoire of respiratory infections, and intimately associated with persistent lung bacterial colonization in patients suffering from chronic obstructive pulmonary disease (COPD). Despite its medical relevance, relatively little is known about its mechanisms of pathogenicity. In this study, we found that NTHi invades the airway epithelium by a distinct mechanism, requiring microtubule assembly, lipid rafts integrity, and activation of phosphatidylinositol 3-kinase (PI3K) signalling. We found that the majority of intracellular bacteria are located inside an acidic subcellular compartment, in a metabolically active and non-proliferative state. This NTHi-containing vacuole (NTHi-CV) is endowed with late endosome features, co-localizing with LysoTracker, lamp-1, lamp-2, CD63 and Rab7. The NTHi-CV does not acquire Golgi- or autophagy-related markers. These observations were extended to immortalized and primary human airway epithelial cells. By using NTHi clinical isolates expressing different amounts of phosphocholine (PCho), a major modification of NTHi lipooligosaccharide, on their surfaces, and an isogenic lic1BC mutant strain lacking PCho, we showed that PCho is not responsible for NTHi intracellular location. In sum, this study indicates that NTHi can survive inside airway epithelial cells.
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Kotnala, Sudhir, Yerin Kim, Charu Rajput, Hymavathi Reddyvari, Sudhir Bolla, Nathaniel T. Marchetti, Beata Kosmider, Karim Bahmed, and Umadevi S. Sajjan. "Contribution of dipeptidyl peptidase 4 to non-typeable Haemophilus influenzae-induced lung inflammation in COPD." Clinical Science 135, no. 17 (September 1, 2021): 2067–83. http://dx.doi.org/10.1042/cs20210099.
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Abstract Dipeptidyl peptidase 4 (DPP4) expression is increased in the lungs of chronic obstructive pulmonary disease (COPD). DPP4 is known to be associated with inflammation in various organs, including LPS-induced acute lung inflammation. Since non-typeable Haemophilus influenzae (NTHi) causes acute exacerbations in COPD patients, we examined the contribution of DPP4 in NTHi-induced lung inflammation in COPD. Pulmonary macrophages isolated from COPD patients showed higher expression of DPP4 than the macrophages isolated from normal subjects. In response to NTHi infection, COPD, but not normal macrophages show a further increase in the expression of DPP4. COPD macrophages also showed higher expression of IL-1β, and CCL3 responses to NTHi than normal, and treatment with DPP4 inhibitor, diprotin A attenuated this response. To examine the contribution of DPP4 in NTHi-induced lung inflammation, COPD mice were infected with NTHi, treated with diprotin A or PBS intraperitoneally, and examined for DPP4 expression, lung inflammation, and cytokine expression. Mice with COPD phenotype showed increased expression of DPP4, which increased further following NTHi infection. DPP4 expression was primarily observed in the infiltrated inflammatory cells. NTHi-infected COPD mice also showed sustained neutrophilic lung inflammation and expression of CCL3, and this was inhibited by DPP4 inhibitor. These observations indicate that enhanced expression of DPP4 in pulmonary macrophages may contribute to sustained lung inflammation in COPD following NTHi infection. Therefore, inhibition of DPP4 may reduce the severity of NTHi-induced lung inflammation in COPD.
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Craig, Jane E., Angela Nobbs, and Nicola J. High. "The Extracytoplasmic Sigma Factor, ςE, Is Required for Intracellular Survival of Nontypeable Haemophilus influenzae in J774 Macrophages." Infection and Immunity 70, no. 2 (February 2002): 708–15. http://dx.doi.org/10.1128/iai.70.2.708-715.2002.
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ABSTRACT Nontypeable Haemophilus influenzae (NTHi) causes a wide variety of respiratory tract infections in humans. It is capable of invading and surviving in epithelial cells and has also been shown to persist in macrophage-like cell line J774A.1. To determine the molecular mechanisms which enable NTHi to survive in an intracellular environment, differential display reverse transcriptase PCR was used to identify genes which were either induced or upregulated by NTHi residing in macrophages. Using this approach, we identified one transcript which was consistently amplified from intracellular NTHi cDNA. Nucleotide sequence analysis of this product revealed that it spanned the 3′ and 5′ ends of rpoE and rseB, respectively, which form part of the extracytoplasmic stress operon that encodes and regulates expression of alternate sigma factor sigma E (ςE). To confirm that expression of rpoE was upregulated following uptake of NTHi by macrophages, an rpoE-lacZ transcriptional fusion was constructed, and expression of β-galactosidase activity in broth-grown NTHi was compared with expression of β-galactosidase activity in intracellular NTHi. The level of β-galactosidase activity in NTHi 4 h after phagocytosis by macrophages was found to be 100-fold higher than that of broth-grown organisms, suggesting that genes of the ςE regulon may be important for persistence of NTHi in mammalian cells. The hypothesis that ςE plays a role in the intracellular survival of NTHi was subsequently confirmed by the decreased ability of an rpoE insertion mutant to survive in macrophages.
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Winter, Linda E., and Stephen J. Barenkamp. "Antibodies Specific for the High-Molecular-Weight Adhesion Proteins of Nontypeable Haemophilus influenzae Are Opsonophagocytic for both Homologous and Heterologous Strains." Clinical and Vaccine Immunology 13, no. 12 (October 4, 2006): 1333–42. http://dx.doi.org/10.1128/cvi.00221-06.
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ABSTRACT The HMW1/HMW2-like adhesion proteins of nontypeable Haemophilus influenzae (NTHI) are expressed by 75% of NTHI strains. Antibodies directed against these proteins are opsonophagocytic in vitro and are protective in an animal model of infection. The objective of the present study was to determine the opsonophagocytic activity of high-titer anti-HMW1/HMW2 immune sera against both homologous and heterologous NTHI strains. Chinchillas were immunized with purified HMW1/HMW2-like proteins from five prototype NTHI strains. Serum opsonophagocytic activity was monitored in an assay that uses a human promyelocytic cell line, HL-60, as the source of phagocytic cells. Preimmune sera did not demonstrate opsonophagocytic killing of any strains. In contrast, the immune sera demonstrated killing of the five homologous NTHI strains at titers ranging from 1:320 to 1:640. The immune sera also demonstrated killing of eight heterologous NTHI strains that express HMW1/HMW2-like proteins at titers ranging from 0 to 1:640. Killing of heterologous strains sometimes demonstrated a prozone phenomenon. None of the immune sera killed NTHI strains that did not express HMW1/HMW2-like proteins. Adsorption of immune sera with HMW1/HMW2-like proteins purified from either homologous or heterologous NTHI strains eliminated opsonophagocytic killing of homologous strains in most cases. These data demonstrate that antibodies produced following immunization with the HMW1/HMW2-like proteins are opsonophagocytic for both homologous and heterologous NTHI and strongly suggest that common epitopes recognized by functionally active antibodies exist on the HMW1/HMW2-like proteins of unrelated NTHI strains. The results argue for the continued investigation of the HMW1/HMW2-like proteins as potential vaccine candidates for the prevention of NTHI disease.
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Armbruster, Chelsie E., Wenzhou Hong, Bing Pang, Kristin E. Dew, Richard A. Juneau, Matthew S. Byrd, Cheraton F. Love, Nancy D. Kock, and W. Edward Swords. "LuxS Promotes Biofilm Maturation and Persistence of Nontypeable Haemophilus influenzae In Vivo via Modulation of Lipooligosaccharides on the Bacterial Surface." Infection and Immunity 77, no. 9 (June 29, 2009): 4081–91. http://dx.doi.org/10.1128/iai.00320-09.
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ABSTRACT Nontypeable Haemophilus influenzae (NTHI) is an extremely common airway commensal which can cause opportunistic infections that are usually localized to airway mucosal surfaces. During many of these infections, NTHI forms biofilm communities that promote persistence in vivo. For many bacterial species, density-dependent quorum-signaling networks can affect biofilm formation and/or maturation. Mutation of luxS, a determinant of the autoinducer 2 (AI-2) quorum signal pathway, increases NTHI virulence in the chinchilla model for otitis media infections. For example, bacterial counts in middle-ear fluids and the severity of the host inflammatory response were increased in luxS mutants compared with parental strains. As these phenotypes are consistent with those that we have observed for biofilm-defective NTHI mutants, we hypothesized that luxS may affect NTHI biofilms. A luxS mutant was generated using the well-characterized NTHI 86-028NP strain and tested to determine the effects of the mutation on biofilm phenotypes in vitro and bacterial persistence and disease severity during experimental otitis media. Quantitation of the biofilm structure by confocal microscopy and COMSTAT analysis revealed significantly reduced biomass for NTHI 86-028NP luxS biofilms, which was restored by a soluble mediator in NTHI 86-028NP supernatants. Analysis of lipooligosaccharide moieties using an enzyme-linked immunosorbent assay and immunoblotting showed decreased levels of biofilm-associated glycoforms in the NTHI 86-028NP luxS strain. Infection studies showed that NTHI 86-028NP luxS had a significant persistence defect in vivo during chronic otitis media infection. Based on these data, we concluded that a luxS-dependent soluble mediator modulates the composition of the NTHI lipooligosaccharides, resulting in effects on biofilm maturation and bacterial persistence in vivo.
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Novotny, Laura, Kenneth Brockman, Elaine Mokrzan, Joseph Jurcisek, and Lauren Bakaletz. "Biofilm Biology and Vaccine Strategies for Otitis Media Due to Nontypeable Haemophilus influenzae." Journal of Pediatric Infectious Diseases 14, no. 02 (July 6, 2018): 069–78. http://dx.doi.org/10.1055/s-0038-1660818.
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AbstractOtitis media (OM) is one of the most common diseases of childhood, and nontypeable Haemophilus influenzae (NTHI) is the predominant causative agent of chronic and recurrent OM, as well as OM for which treatment has failed. Moreover, NTHI is now as important a causative agent of acute OM as the pneumococcus. NTHI colonizes the human nasopharynx asymptomatically. However, upon perturbation of the innate and physical defenses of the airway by upper respiratory tract viral infection, NTHI can replicate, ascend the Eustachian tube, gain access to the normally sterile middle ear space, and cause disease. Bacterial biofilms within the middle ear, including those formed by NTHI, contribute to the chronic and recurrent nature of this disease. These multicomponent structures are highly resistant to clearance by host defenses and elimination by traditional antimicrobial therapies. Herein, we review several strategies utilized by NTHI to persist within the human host and interventions currently under investigation to prevent and/or resolve NTHI-induced diseases of the middle ear and uppermost airway.
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Juneau, Richard A., Bing Pang, Chelsie E. Armbruster, Kyle A. Murrah, Antonia C. Perez, and W. Edward Swords. "Peroxiredoxin-Glutaredoxin and Catalase Promote Resistance of Nontypeable Haemophilus influenzae 86-028NP to Oxidants and Survival within Neutrophil Extracellular Traps." Infection and Immunity 83, no. 1 (October 27, 2014): 239–46. http://dx.doi.org/10.1128/iai.02390-14.
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NontypeableHaemophilus influenzae(NTHI) is a common commensal and opportunistic pathogen of the human airways. For example, NTHI is a leading cause of otitis media and is the most common cause of airway infections associated with chronic obstructive pulmonary disease (COPD). These infections are often chronic/recurrent in nature and involve bacterial persistence within biofilm communities that are highly resistant to host clearance. Our previous work has shown that NTHI within biofilms has increased expression of factors associated with oxidative stress responses. The goal of this study was to define the roles of catalase (encoded byhktE) and a bifunctional peroxiredoxin-glutaredoxin (encoded bypdgX) in resistance of NTHI to oxidants and persistencein vivo. Isogenic NTHI strain 86-028NP mutants lackinghktEandpdgXhad increased susceptibility to peroxide. Moreover, these strains had persistence defects in the chinchilla infection model for otitis media, as well as in a murine model for COPD. Additional work showed thatpdgXandhktEwere important determinants of NTHI survival within neutrophil extracellular traps (NETs), which we have shown to be an integral part of NTHI biofilmsin vivo. Based on these data, we conclude that catalase and peroxiredoxin-glutaredoxin are determinants of bacterial persistence during chronic/recurrent NTHI infections that promote bacterial survival within NETs.
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Clementi, Cara F., Anders P. Håkansson, and Timothy F. Murphy. "Internalization and Trafficking of Nontypeable Haemophilus influenzae in Human Respiratory Epithelial Cells and Roles of IgA1 Proteases for Optimal Invasion and Persistence." Infection and Immunity 82, no. 1 (November 11, 2013): 433–44. http://dx.doi.org/10.1128/iai.00864-13.
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ABSTRACTNontypeableHaemophilus influenzae(NTHI) is a leading cause of opportunistic infections of the respiratory tract in children and adults. Although considered an extracellular pathogen, NTHI has been observed repeatedly within and between cells of the human respiratory tract, and these observations have been correlated to symptomatic infection. These findings are intriguing in light of the knowledge that NTHI persists in the respiratory tract despite antibiotic therapy and the development of bactericidal antibodies. We hypothesized that intracellular NTHI avoids, escapes, or neutralizes the endolysosomal pathway and persists within human respiratory epithelial cells and that human IgA1 proteases are required for optimal internalization and persistence of NTHI. Virtually all strains encode a human IgA1 protease gene,igaA, and we previously characterized a novel human IgA1 protease gene,igaB, that is associated with disease-causing strains and is homologous to the IgA1 protease that is unique to pathogenicNeisseriaspp. Here, we show that NTHI invades human bronchial epithelial cellsin vitroin a lipid raft-independent manner, is subsequently trafficked via the endolysosomal pathway, and is killed in lysosomes after variable durations of persistence. IgaA is required for optimal invasion. IgaB appears to play little or no role in adherence or invasion but is required for optimal intracellular persistence of NTHI. IgaB cleaves lysosome-associated membrane protein 1 (LAMP1) at pHs characteristic of the plasma membrane, early endosome, late endosome, and lysosome. However, neither IgA1 protease inhibits acidification of intracellular vesicles containing NTHI. NTHI IgA1 proteases play important but different roles in NTHI invasion and trafficking in respiratory epithelial cells.
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Mokrzan, Elaine M., Michael O. Ward, and Lauren O. Bakaletz. "Type IV Pilus Expression Is Upregulated in Nontypeable Haemophilus influenzae Biofilms Formed at the Temperature of the Human Nasopharynx." Journal of Bacteriology 198, no. 19 (April 4, 2016): 2619–30. http://dx.doi.org/10.1128/jb.01022-15.
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ABSTRACTNontypeableHaemophilus influenzae(NTHI), a commensal of the human nasopharynx (hNP), is a common cause of biofilm-associated diseases of the respiratory tract. However, NTHI biofilm biology at the average hNP temperature, i.e., 34°C, has not been well studied. Here we grew NTHI biofilms at 34°C and 37°C, to evaluate relative biofilm growth, expression, and function of the type IV pilus (Tfp), a critical adhesin important for NTHI biofilm formation. The kinetics and regulation of Tfp expression in NTHI biofilms are unclear, especially at 34°C. Tfp expression, as estimated bypilApromoter activity, was distributed throughout the biofilms, with a unique pattern that was dependent on temperature, time in culture, and position within the maturing biofilm. Tfp expression was required for the formation of the characteristic tower structures of NTHI biofilms and was significantly upregulated in NTHI biofilms formed at 34°C versus 37°C. This increase correlated with significantly greater twitching motility at 34°C than at 37°C. Treatment with antisera targeting the major subunit of Tfp (PilA) significantly inhibited NTHI biofilm formation at both temperatures, confirming the importance of this critical adhesin in biofilm formation. Additionally, treatment of preestablished biofilms with antisera against PilA significantly decreased biofilm biomass and mean thickness at both temperatures. These results demonstrated a pivotal role for Tfp in NTHI biofilm formation and stability at the temperature of the hNP, and they underscore the utility of PilA as a vaccine candidate for treatment and/or prevention of NTHI biofilm-associated diseases.IMPORTANCENTHI is an important cause of chronic respiratory tract infections, including otitis media, chronic rhinosinusitis, and exacerbations of chronic obstructive pulmonary disease and cystic fibrosis. The chronic and recurrent nature of these diseases is attributed to the presence of bacterial biofilms, which are highly resistant to antimicrobials. We characterized NTHI biofilm growth and expression of PilA, the major subunit of the Tfp, at the temperature of the hNP, which is the commensal habitat of NTHI. Our results expand the current understanding of the role of Tfp during biofilm formation and maturation at the temperature of both the hNP and the middle ear, and they strengthen support for PilA as a vaccine candidate for the prevention and treatment of NTHI biofilm-associated diseases.
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Atto, Brianna, Dale Kunde, David A. Gell, and Stephen Tristram. "Oropharyngeal Carriage of hpl-Containing Haemophilus haemolyticus Predicts Lower Prevalence and Density of NTHi Colonisation in Healthy Adults." Pathogens 10, no. 5 (May 10, 2021): 577. http://dx.doi.org/10.3390/pathogens10050577.
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Nontypeable Haemophilus influenzae (NTHi) is a major respiratory pathogen that initiates infection by colonising the upper airways. Strategies that interfere with this interaction may therefore have a clinically significant impact on the ability of NTHi to cause disease. We have previously shown that strains of the commensal bacterium Haemophilus haemolyticus (Hh) that produce a novel haem-binding protein, haemophilin, can prevent NTHi growth and interactions with host cells in vitro. We hypothesized that natural pharyngeal carriage of Hh strains with the hpl open reading frame (Hh-hpl+) would be associated with a lower prevalence and/or density of NTHi colonisation in healthy individuals. Oropharyngeal swabs were collected from 257 healthy adults in Australia between 2018 and 2019. Real-time PCR was used to quantitatively compare the oropharyngeal carriage load of NTHi and Hh populations with the Hh-hpl+ or Hh-hpl− genotype. The likelihood of acquiring/maintaining NTHi colonisation status over a two- to six-month period was assessed in individuals that carried either Hh-hpl− (n = 25) or Hh-hpl+ (n = 25). Compared to carriage of Hh-hpl− strains, adult (18–65 years) and elderly (>65 years) participants that were colonised with Hh-hpl+ were 2.43 or 2.67 times less likely to carry NTHi in their oropharynx, respectively. Colonisation with high densities of Hh-hpl+ correlated with a low NTHi carriage load and a 2.63 times lower likelihood of acquiring/maintaining NTHi colonisation status between visits. Together with supporting in vitro studies, these results encourage further investigation into the potential use of Hh-hpl+ as a respiratory probiotic candidate for the prevention of NTHi infection.
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Winter, Linda E., and Stephen J. Barenkamp. "Antibodies to the HMW1/HMW2 and Hia Adhesins of Nontypeable Haemophilus influenzae Mediate Broad-Based Opsonophagocytic Killing of Homologous and Heterologous Strains." Clinical and Vaccine Immunology 21, no. 5 (February 26, 2014): 613–21. http://dx.doi.org/10.1128/cvi.00772-13.
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ABSTRACTThe HMW1/HMW2 and Hia proteins are highly immunogenic surface adhesins of nontypeableHaemophilus 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 fromEscherichia colitransformants. 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.
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West-Barnette, Shayla, Andrea Rockel, and W. Edward Swords. "Biofilm Growth Increases Phosphorylcholine Content and Decreases Potency of Nontypeable Haemophilus influenzae Endotoxins." Infection and Immunity 74, no. 3 (March 2006): 1828–36. http://dx.doi.org/10.1128/iai.74.3.1828-1836.2006.
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ABSTRACT Nontypeable Haemophilus influenzae (NTHI) is a common respiratory commensal and opportunistic pathogen. NTHI is normally contained within the airways by host innate defenses that include recognition of bacterial endotoxins by Toll-like receptor 4 (TLR4). NTHI produces lipooligosaccharide (LOS) endotoxins which lack polymeric O side chains and which may contain host glycolipids. We recently showed that NTHI biofilms contain variants with sialylated LOS glycoforms that are essential to biofilm formation. In this study, we show that NTHI forms biofilms on epithelial cell layers. Confocal analysis revealed that sialylated variants were distributed throughout the biofilm, while variants expressing phosphorylcholine (PCho) were found within the biofilm. Consistent with this observation, PCho content of LOS purified from NTHI biofilms was increased compared to LOS from planktonic cultures. Hypothesizing that the observed changes in endotoxin composition could affect bioactivity, we compared inflammatory responses to NTHI LOS purified from biofilm and planktonic cultures. Our results show that endotoxins from biofilms induced weaker host innate responses. While we observed a minimal effect of sialylation on LOS bioactivity, there was a significant decrease in bioactivity associated with PCho substitutions. We thus conclude that biofilm growth increases the proportion of PCho+ variants in an NTHI population, resulting in a net decrease in LOS bioactivity. Thus, in addition to their well-documented resistance phenotypes, our data show that biofilm communities of NTHI bacteria contain variants that evoke less potent host responses.
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Wegele, Christian, Carolin Stump-Guthier, Selina Moroniak, Christel Weiss, Manfred Rohde, Hiroshi Ishikawa, Horst Schroten, Christian Schwerk, Michael Karremann, and Julia Borkowski. "Non-Typeable Haemophilus influenzae Invade Choroid Plexus Epithelial Cells in a Polar Fashion." International Journal of Molecular Sciences 21, no. 16 (August 10, 2020): 5739. http://dx.doi.org/10.3390/ijms21165739.
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Non-typeable Haemophilus influenzae (NTHI) is a pathogen of the human respiratory tract causing the majority of invasive H. influenzae infections. Severe invasive infections such as septicemia and meningitis occur rarely, but the lack of a protecting vaccine and the increasing antibiotic resistance of NTHI impede treatment and emphasize its relevance as a potential meningitis causing pathogen. Meningitis results from pathogens crossing blood–brain barriers and invading the immune privileged central nervous system (CNS). In this study, we addressed the potential of NTHI to enter the brain by invading cells of the choroid plexus (CP) prior to meningeal inflammation to enlighten NTHI pathophysiological mechanisms. A cell culture model of human CP epithelial cells, which form the blood–cerebrospinal fluid barrier (BCSFB) in vivo, was used to analyze adhesion and invasion by immunofluorescence and electron microscopy. NTHI invade CP cells in vitro in a polar fashion from the blood-facing side. Furthermore, NTHI invasion rates are increased compared to encapsulated HiB and HiF strains. Fimbriae occurrence attenuated adhesion and invasion. Thus, our findings underline the role of the BCSFB as a potential entry port for NTHI into the brain and provide strong evidence for a function of the CP during NTHI invasion into the CNS during the course of meningitis.
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Wieland, Catharina W., Sandrine Florquin, and Tom van der Poll. "Interleukin 18 Participates in the Early Inflammatory Response and Bacterial Clearance during Pneumonia Caused by Nontypeable Haemophilus influenzae." Infection and Immunity 75, no. 10 (July 30, 2007): 5068–72. http://dx.doi.org/10.1128/iai.00287-07.
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ABSTRACT Nontypeable Haemophilus influenzae (NTHi) is a common gram-negative respiratory pathogen. To determine the role of the proinflammatory cytokine interleukin 18 (IL-18) during NTHi pneumonia, normal wild-type (WT) and IL-18 knockout (KO) mice were intranasally infected with NTHi. IL-18 KO mice displayed a delayed clearance of NTHi from the respiratory tract, resulting in >20-fold higher bacterial loads in their lungs at 24 h after infection, preceded by a strongly attenuated pulmonary innate immune response as determined by cytokine and chemokine induction and histopathology. These data identify IL-18 as part of an adequate innate immune response during NTHi pneumonia.
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Hong, Wenzhou, Kevin Mason, Joseph Jurcisek, Laura Novotny, Lauren O. Bakaletz, and W. Edward Swords. "Phosphorylcholine Decreases Early Inflammation and Promotes the Establishment of Stable Biofilm Communities of Nontypeable Haemophilus influenzae Strain 86-028NP in a Chinchilla Model of Otitis Media." Infection and Immunity 75, no. 2 (November 27, 2006): 958–65. http://dx.doi.org/10.1128/iai.01691-06.
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ABSTRACT Nontypeable Haemophilus influenzae (NTHi) is a leading causative agent of otitis media. Much of the inflammation occurring during NTHi disease is initiated by lipooligosaccharides (LOS) on the bacterial surface. Phosphorylcholine (PCho) is added to some LOS forms in a phase-variable manner, and these PCho+ variants predominate in vivo. Thus, we asked whether this modification confers some advantage during infection. Virulence of an otitis media isolate (NTHi strain 86-028NP) was compared with that of an isogenic PCho transferase (licD) mutant using a chinchilla (Chinchilla lanigera) model of otitis media. Animals infected with NTHi 86-028NP licD demonstrated increased early inflammation and a delayed increase in bacterial counts compared to animals infected with NTHi 86-028NP. LOS purified from chinchilla-passed NTHi 86-028NP had increased PCho content compared to LOS purified from the inoculum. Both strains were recovered from middle ear fluids as long as 14 days postinfection. Biofilms were macroscopically visible in the middle ears of euthanized animals infected with NTHi 86-028NP 7 days and 14 days postchallenge. Conversely, less dense biofilms were observed in animals infected with NTHi 86-028NP licD 7 days postinfection, and none of the animals infected with NTHi 86-028NP licD had a visible biofilm by 14 days. Fluorescent antibody staining revealed PCho+ variants within biofilms, similar to our prior results with tissue culture cells in vitro (S. L. West-Barnette, A. Rockel, and W. E. Swords, Infect. Immun. 74:1828-1836, 2006). Animals coinfected with equal proportions of both strains had equal persistence of each strain and somewhat greater severity of disease. We thus conclude that PCho promotes NTHi infection and persistence by reducing the host inflammatory response and by promoting formation of stable biofilm communities.
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Webb, Dianne C., and Allan W. Cripps. "Immunization with Recombinant Transferrin Binding Protein B Enhances Clearance of Nontypeable Haemophilus influenzae from the Rat Lung." Infection and Immunity 67, no. 5 (May 1, 1999): 2138–44. http://dx.doi.org/10.1128/iai.67.5.2138-2144.1999.
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ABSTRACT Nontypeable Haemophilus influenzae (NTHI) is an opportunistic pathogen, and heterogeneity in the surface-exposed immunodominant domains of NTHI proteins is thought to be associated with the failure of an infection to stimulate an immune response that is cross-protective against heterologous NTHI strains. The aim of this study was to assess the vaccine potential of a surface-exposed component of the NTHI human transferrin receptor, TbpB, and to determine if the antibody response elicited was cross-reactive with heterologous strains of NTHI. The efficacy of immunization with a recombinant form of TbpB (rTbpB) was determined by assessing the pulmonary clearance of viable bacteria 4 h after a live challenge with NTHI. There was a significant reduction in the number of viable bacteria in both the bronchoalveolar lavage fluid (34% for the 20-μg dose and 58% for the 40-μg dose) and lung homogenates (26% for the 20-μg dose and 60% for the 40-μg dose) of rats immunized with rTbpB compared to the control animals. While rTbpB-specific antibodies from immunized rats were nonspecific in the recognition of TbpB from six heterologous NTHI strains on Western blots, these antibodies differed in their ability to block transferrin binding to heterologous strains and to cross-react in bactericidal assays. If bactericidal antibodies are key indicators of the efficacy of the immune response in eliminating NTHI, this data suggests that while immunization with rTbpB stimulates protective responses against the homologous isolate, variability in the recognition of TbpB from heterologous isolates may limit the potential of rTbpB as an NTHI vaccine component.
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Euba, Begoña, Javier Moleres, Víctor Segura, Cristina Viadas, Pau Morey, David Moranta, José Leiva, Juan Pablo de-Torres, José Antonio Bengoechea, and Junkal Garmendia. "Genome Expression Profiling-Based Identification and Administration Efficacy of Host-Directed Antimicrobial Drugs against Respiratory Infection by Nontypeable Haemophilus influenzae." Antimicrobial Agents and Chemotherapy 59, no. 12 (September 28, 2015): 7581–92. http://dx.doi.org/10.1128/aac.01278-15.
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ABSTRACTTherapies that are safe, effective, and not vulnerable to developing resistance are highly desirable to counteract bacterial infections. Host-directed therapeutics is an antimicrobial approach alternative to conventional antibiotics based on perturbing host pathways subverted by pathogens during their life cycle by using host-directed drugs. In this study, we identified and evaluated the efficacy of a panel of host-directed drugs against respiratory infection by nontypeableHaemophilus influenzae(NTHi). NTHi is an opportunistic pathogen that is an important cause of exacerbation of chronic obstructive pulmonary disease (COPD). We screened for host genes differentially expressed upon infection by the clinical isolate NTHi375 by analyzing cell whole-genome expression profiling and identified a repertoire of host target candidates that were pharmacologically modulated. Based on the proposed relationship between NTHi intracellular location and persistence, we hypothesized that drugs perturbing host pathways used by NTHi to enter epithelial cells could have antimicrobial potential against NTHi infection. Interfering drugs were tested for their effects on bacterial and cellular viability, on NTHi-epithelial cell interplay, and on mouse pulmonary infection. Glucocorticoids and statins lackedin vitroand/orin vivoefficacy. Conversely, the sirtuin-1 activator resveratrol showed a bactericidal effect against NTHi, and the PDE4 inhibitor rolipram showed therapeutic efficacy by lowering NTHi375 counts intracellularly and in the lungs of infected mice. PDE4 inhibition is currently prescribed in COPD, and resveratrol is an attractive geroprotector for COPD treatment. Together, these results expand our knowledge of NTHi-triggered host subversion and frame the antimicrobial potential of rolipram and resveratrol against NTHi respiratory infection.
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Erwin, Alice L., Sara A. Sandstedt, Paul J. Bonthuis, Jennifer L. Geelhood, Kevin L. Nelson, William C. T. Unrath, Mathew A. Diggle, et al. "Analysis of Genetic Relatedness of Haemophilus influenzae Isolates by Multilocus Sequence Typing." Journal of Bacteriology 190, no. 4 (December 7, 2007): 1473–83. http://dx.doi.org/10.1128/jb.01207-07.
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ABSTRACT The gram-negative bacterium Haemophilus influenzae is a human-restricted commensal of the nasopharynx that can also be associated with disease. The majority of H. influenzae respiratory isolates lack the genes for capsule production and are nontypeable (NTHI). Whereas encapsulated strains are known to belong to serotype-specific phylogenetic groups, the structure of the NTHI population has not been previously described. A total of 656 H. influenzae strains, including 322 NTHI strains, have been typed by multilocus sequence typing and found to have 359 sequence types (ST). We performed maximum-parsimony analysis of the 359 sequences and calculated the majority-rule consensus of 4,545 resulting equally most parsimonious trees. Eleven clades were identified, consisting of six or more ST on a branch that was present in 100% of trees. Two additional clades were defined by branches present in 91% and 82% of trees, respectively. Of these 13 clades, 8 consisted predominantly of NTHI strains, three were serotype specific, and 2 contained distinct NTHI-specific and serotype-specific clusters of strains. Sixty percent of NTHI strains have ST within one of the 13 clades, and eBURST analysis identified an additional phylogenetic group that contained 20% of NTHI strains. There was concordant clustering of certain metabolic reactions and putative virulence loci but not of disease source or geographic origin. We conclude that well-defined phylogenetic groups of NTHI strains exist and that these groups differ in genetic content. These observations will provide a framework for further study of the effect of genetic diversity on the interaction of NTHI with the host.
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Megantara, Imam, Ronny Lesmana, Nova Sylviana, Sunarjati Soedigdoadi, and Teti Madiadipoera. "The Potency of NTHi lic1A Gene as a Biomarker in Determining The Severity of Post-Viral Acute Rhinosinusitis." Indonesian Biomedical Journal 13, no. 3 (September 9, 2021): 303–9. http://dx.doi.org/10.18585/inabj.v13i3.1614.
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BACKGROUND: The lic1A gene is an important virulence factor for non-typeable H. influenzae (NTHi), which allows its translocation from the nasopharynx into the sinonasal cavity and modulates more severe inflammatory processes. This study is aimed for identifying the potential correlation between the NTHi lic1A gene expressions and the severity of post-viral acute rhinosinusitis.METHODS: Sixty patients who were diagnosed with post-viral acute rhinosinusitis, were recruited from an ENT clinic in a referral hospital, in Bandung, West Java, Indonesia. All patients underwent a historical assessment and ENT examination. The nasal specimen was taken from the patient’s middle meatal. The NTHi lic1A gene expression was detected using Polymerase Chain Reaction (PCR).RESULTS: We observed that eight patients had the NTHi lic1A (+), with a strong correlation toward the dominant symptoms (nasal obstruction and discharge). In addition, the symptom’s duration of the NTHi lic1A (+) was twice longer than patients with the NTHi lic1A (-). Its severity was significantly more different between the two groups (p=0.034).CONCLUSION: Taken together, the presence of the NTHi lic1A gene is significantly associated with the severity of the disease and the symptom’s duration. Thus, the NTHi lic1A gene could potentially be a good marker for assessing the severity of post-viral acute rhinosinusitis cases in the future.KEYWORDS: H. influenzae, rhinosinusitis, nasal obstruction, virulence factors
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Thofte, Oskar, Emma Mattsson, Yu Ching Su, John Thegerström, Farshid Jalalvand, Anna Marie Blom, and Kristian Riesbeck. "Non-typeable Haemophilus influenzae P5 binds C4b-binding protein promoting serum resistance." Journal of Immunology 204, no. 1_Supplement (May 1, 2020): 82.23. http://dx.doi.org/10.4049/jimmunol.204.supp.82.23.
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Abstract Haemophilus influenzae is a Gram-negative bacterium considered as a commensal in pre-school children, mainly giving rise to infections locally in the upper and lower respiratory tract. Historically invasive disease has been caused by encapsulated strains, mainly H. influenzae serotype b (Hib). However, since introduction of the Hib vaccine, there has been an increased incidence of invasive disease with unencapsulated non-typeable H. influenzae (NTHi). Since NTHi lacks a capsule they must rely on other mechanisms for serum resistance, including binding of complement regulators. We have previously demonstrated that binding of C4b-binding protein (C4BP) is important for NTHi-dependent complement evasion. Here we identified outer membrane protein 5 (P5) as the C4BP-binding protein in NTHi based on an outer membrane proteomic pull down-assay. NTHi P5 has previously been found to bind factor H, an inhibitor of the alternative complement pathway. We found that NTHi grown in nasopharyngeal-simulated conditions has a lower surface expression of P5. This was also corresponding to decreased serum resistance. Importantly, we found that P5-deficient NTHi mutants showed significantly decreased C4BP binding and serum resistance. Additionally, surface expression of P5 on Escherichia coli conferred increased serum resistance and C4BP binding. Our results further highlights P5 as an important protein for protecting NTHi against complement-mediated killing.
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Post, Deborah M. B., Margaret R. Ketterer, Jeremy E. Coffin, Lorri M. Reinders, Robert S. Munson, Thomas Bair, Timothy F. Murphy, Eric D. Foster, Bradford W. Gibson, and Michael A. Apicella. "Comparative Analyses of the Lipooligosaccharides from Nontypeable Haemophilus influenzae and Haemophilus haemolyticus Show Differences in Sialic Acid and Phosphorylcholine Modifications." Infection and Immunity 84, no. 3 (January 4, 2016): 765–74. http://dx.doi.org/10.1128/iai.01185-15.
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Haemophilus haemolyticusand nontypeableHaemophilus influenzae(NTHi) are closely related upper airway commensal bacteria that are difficult to distinguish phenotypically. NTHi causes upper and lower airway tract infections in individuals with compromised airways, whileH. haemolyticusrarely causes such infections. The lipooligosaccharide (LOS) is an outer membrane component of both species and plays a role in NTHi pathogenesis. In this study, comparative analyses of the LOS structures and corresponding biosynthesis genes were performed. Mass spectrometric and immunochemical analyses showed that NTHi LOS contained terminal sialic acid more frequently and to a higher extent thanH. haemolyticusLOS did. Genomic analyses of 10 strains demonstrated thatH. haemolyticuslacked the sialyltransferase geneslic3Aandlic3B(9/10) andsiaA(10/10), but all strains contained the sialic acid uptake genessiaPandsiaT(10/10). However, isothermal titration calorimetry analyses of SiaP from twoH. haemolyticusstrains showed a 3.4- to 7.3-fold lower affinity for sialic acid compared to that of NTHi SiaP. Additionally, mass spectrometric and immunochemical analyses showed that the LOS fromH. haemolyticuscontained phosphorylcholine (ChoP) less frequently than the LOS from NTHi strains. These differences observed in the levels of sialic acid and ChoP incorporation in the LOS structures fromH. haemolyticusand NTHi may explain some of the differences in their propensities to cause disease.
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Clemans, Daniel L., Richard J. Bauer, Julie A. Hanson, Monte V. Hobbs, Joseph W. St. Geme, Carl F. Marrs, and Janet R. Gilsdorf. "Induction of Proinflammatory Cytokines from Human Respiratory Epithelial Cells after Stimulation by NontypeableHaemophilus influenzae." Infection and Immunity 68, no. 8 (August 1, 2000): 4430–40. http://dx.doi.org/10.1128/iai.68.8.4430-4440.2000.
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ABSTRACT Nontypeable Haemophilus influenzae (NTHi) causes repeated respiratory infections in patients with chronic lung diseases. These infections are characterized by a brisk inflammatory response which results in the accumulation of polymorphonucleated cells in the lungs and is dependent on the expression and secretion of proinflammatory cytokines. We hypothesize that multiple NTHi molecules, including lipooligosaccharide (LOS), mediate cellular interactions with respiratory epithelial cells, leading to the production of proinflammatory cytokines. To address this hypothesis, we exposed 9HTEo− human tracheal epithelial cells to NTHi and compared the resulting profiles of cytokine gene expression and secretion using multiprobe RNase protection assays and enzyme-linked immunosorbent assays (ELISA), respectively. Dose-response experiments demonstrated a maximum stimulation of most cytokines tested, using a ratio of 100 NTHi bacterial cells to 1 9HTEo− tracheal epithelial cell. Compared with purified LOS, NTHi bacterial cells stimulated 3.6- and 4.5-fold increases in epithelial cell expression of interleukin-8 (IL-8) and IL-6 genes, respectively. Similar results were seen with epithelial cell macrophage chemotactic protein 1, IL-1α, IL-1β, and tumor necrosis factor alpha expression. Polymyxin B completely inhibited LOS stimulation but only partially reduced NTHi whole cell stimulation. Taken together, these results suggest that multiple bacterial molecules including LOS contribute to the NTHi stimulation of respiratory epithelial cell cytokine production. Moreover, no correlation was seen between NTHi adherence to epithelial cells mediated by hemagglutinating pili, Hia, HMW1, HMW2, and Hap and epithelial cytokine secretion. These data suggest that bacterial molecules beyond previously described NTHi cell surface adhesins and LOS play a role in the induction of proinflammatory cytokines from respiratory epithelial cells.
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Smith-Vaughan, Heidi C., Anne B. Chang, Derek S. Sarovich, Robyn L. Marsh, Keith Grimwood, Amanda J. Leach, Peter S. Morris, and Erin P. Price. "Absence of an Important Vaccine and Diagnostic Target in Carriage- and Disease-Related Nontypeable Haemophilus influenzae." Clinical and Vaccine Immunology 21, no. 2 (November 27, 2013): 250–52. http://dx.doi.org/10.1128/cvi.00632-13.
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ABSTRACTNontypeableHaemophilus influenzae(NTHi)-associated disease is a major health problem globally. Whole-genome sequence analysis identified the absence ofhpdgenes encodingHaemophilusprotein D in 3 of 16 phylogenetically distinct NTHi isolates. This novel finding is of potential clinical significance, as protein D andhpdrepresent important NTHi vaccine antigen and diagnostic targets, respectively.
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Raffel, Forrest K., Blake R. Szelestey, Wandy L. Beatty, and Kevin M. Mason. "The Haemophilus influenzae Sap Transporter Mediates Bacterium-Epithelial Cell Homeostasis." Infection and Immunity 81, no. 1 (October 15, 2012): 43–54. http://dx.doi.org/10.1128/iai.00942-12.
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NontypeableHaemophilus influenzae(NTHI) is a commensal inhabitant of the human nasopharynx and a causative agent of otitis media and other diseases of the upper and lower human airway. During colonization within the host, NTHI must acquire essential nutrients and evade immune attack. We previously demonstrated that the NTHI Sap transporter, an inner membrane protein complex, mediates resistance to antimicrobial peptides and is required for heme homeostasis. We hypothesized that Sap transporter functions are critical for NTHI interaction with the host epithelium and establishment of colonization. Thus, we cocultured the parent or thesapAmutant on polarized epithelial cells grown at an air-liquid interface, as a physiological model of NTHI colonization, to determine the contribution of the Sap transporter to bacterium-host cell interactions. Although SapA-deficient NTHI was less adherent to epithelial cells, we observed a significant increase in invasive bacteria compared to the parent strain. Upon internalization, thesapAmutant appeared free in the cytoplasm, whereas the parent strain was primarily found in endosomes, indicating differential subcellular trafficking. Additionally, we observed reduced inflammatory cytokine production by the epithelium in response to thesapAmutant strain compared to the parental strain. Furthermore, chinchilla middle ears challenged with thesapAmutant demonstrated a decrease in disease severity compared to ears challenged with the parental strain. Collectively, our data suggest that NTHI senses host environmental cues via Sap transporter function to mediate interaction with host epithelial cells. Epithelial cell invasion and modulation of host inflammatory cytokine responses may promote NTHI colonization and access to essential nutrients.
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Kennedy, Bobbie-Jo, Laura A. Novotny, Joseph A. Jurcisek, Yves Lobet, and Lauren O. Bakaletz. "Passive Transfer of Antiserum Specific for Immunogens Derived from a Nontypeable Haemophilus influenzaeAdhesin and Lipoprotein D Prevents Otitis Media after Heterologous Challenge." Infection and Immunity 68, no. 5 (May 1, 2000): 2756–65. http://dx.doi.org/10.1128/iai.68.5.2756-2765.2000.
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ABSTRACT We recently determined that passive transfer of serum directed against a synthetic peptide called LB1 or a recombinant fusion protein immunogen [LPD-LB1(f)2,1,3] could prevent otitis media after challenge with a homologous nontypeable Haemophilus influenzae (NTHI) isolate. NTHI residing in the nasopharynx was rapidly cleared from this site, thus preventing it from ascending the eustachian tube and inducing otitis media in chinchillas compromised by an ongoing viral upper respiratory tract infection. While LB1 is based solely on one NTHI adhesin, the latter immunogen, LPD-LB1(f)2,1,3, was designed to incorporate two NTHI antigens shown to play a role in the pathogenesis of otitis media; lipoprotein D (LPD) and the P5-homologous fimbrin adhesin. The design of LPD-LB1(f)2,1,3 also accommodated for the recently demonstrated existence of three major groupings, based on amino acid sequence diversity, in the third surface-exposed region of P5-fimbrin. LPD-LB1(f)2,1,3 was thus designed to potentially confer broader protection against challenge by diverse strains of NTHI. Chinchillas were passively immunized here with serum specific for either LB1 or for LPD-LB1(f)2,1,3 prior to challenge with a member of all three groups of NTHI relative to diversity in region 3. The transferred serum pools were also analyzed for titer, specificity, and several functional activities. We found that both serum pools had equivalent ability to mediate C′-dependent killing and to inhibit adherence of NTHI strains to human oropharyngeal cells. When passively transferred, both serum pools significantly inhibited the signs and incidence of otitis media (P ≤ 0.01) induced by any of the three challenge isolates. Despite providing protection against disease, the ability of these antisera to induce total eradication of NTHI from the nasopharynx was not equivalent among NTHI groups. These data thus suggested that while early, complete eradication of NTHI from the nasopharynx was highly protective, reduction of the bacterial load to below a critical threshold level appeared to be similarly effective.
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Fernández-Calvet, Ariadna, Begoña Euba, Lucía Caballero, Roberto Díez-Martínez, Margarita Menéndez, Carlos Ortiz de Solórzano, José Leiva, Vicente Micol, Enrique Barrajón-Catalán, and Junkal Garmendia. "Preclinical Evaluation of the Antimicrobial-Immunomodulatory Dual Action of Xenohormetic Molecules against Haemophilus influenzae Respiratory Infection." Biomolecules 9, no. 12 (December 17, 2019): 891. http://dx.doi.org/10.3390/biom9120891.
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Chronic obstructive pulmonary disease (COPD) is characterized by abnormal inflammation and impaired airway immunity, providing an opportunistic platform for nontypeable Haemophilus influenzae (NTHi) infection. In this context, therapies targeting not only overactive inflammation without significant adverse effects, but also infection are of interest. Increasing evidence suggests that polyphenols, plant secondary metabolites with anti-inflammatory and antimicrobial properties, may be protective. Here, a Cistus salviifolius plant extract containing quercetin, myricetin, and punicalagin was shown to reduce NTHi viability. Analysis of these polyphenols revealed that quercetin has a bactericidal effect on NTHi, does not display synergies, and that bacteria do not seem to develop resistance. Moreover, quercetin lowered NTHi airway epithelial invasion through a mechanism likely involving inhibition of Akt phosphorylation, and reduced the expression of bacterially-induced proinflammatory markers il-8, cxcl-1, il-6, pde4b, and tnfα. We further tested quercetin’s effect on NTHi murine pulmonary infection, showing a moderate reduction in bacterial counts and significantly reduced expression of proinflammatory genes, compared to untreated mice. Quercetin administration during NTHi infection on a zebrafish septicemia infection model system showed a bacterial clearing effect without signs of host toxicity. In conclusion, this study highlights the therapeutic potential of the xenohormetic molecule quercetin against NTHi infection.
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Swords, W. Edward, Miranda L. Moore, Luciana Godzicki, Gail Bukofzer, Michael J. Mitten, and Jessica VonCannon. "Sialylation of Lipooligosaccharides Promotes Biofilm Formation by Nontypeable Haemophilus influenzae." Infection and Immunity 72, no. 1 (January 2004): 106–13. http://dx.doi.org/10.1128/iai.72.1.106-113.2004.
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ABSTRACT Nontypeable Haemophilus influenzae (NTHi) is a major cause of opportunistic respiratory tract infections, including otitis media and bronchitis. The persistence of NTHi in vivo is thought to involve bacterial persistence in a biofilm community. Therefore, there is a need for further definition of bacterial factors contributing to biofilm formation by NTHi. Like other bacteria inhabiting host mucosal surfaces, NTHi has on its surface a diverse array of lipooligosaccharides (LOS) that influence host-bacterial interactions. In this study, we show that LOS containing sialic (N-acetyl-neuraminic) acid promotes biofilm formation by NTHi in vitro and bacterial persistence within the middle ear or lung in vivo. LOS from NTHi in biofilms was sialylated, as determined by comparison of electrophoretic mobilities and immunochemical reactivities before and after neuraminidase treatment. Biofilm formation was significantly reduced in media lacking sialic acid, and a siaB (CMP-sialic acid synthetase) mutant was deficient in biofilm formation in three different in vitro model systems. The persistence of an asialylated siaB mutant was attenuated in a gerbil middle ear infection model system, as well as in a rat pulmonary challenge model system. These data show that sialylated LOS glycoforms promote biofilm formation by NTHi and persistence in vivo.
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Hong, Wenzhou, Bing Pang, Shayla West-Barnette, and W. Edward Swords. "Phosphorylcholine Expression by Nontypeable Haemophilus influenzae Correlates with Maturation of Biofilm Communities In Vitro and In Vivo." Journal of Bacteriology 189, no. 22 (June 15, 2007): 8300–8307. http://dx.doi.org/10.1128/jb.00532-07.
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ABSTRACT Nontypeable Haemophilus influenzae (NTHI) causes chronic infections that feature the formation of biofilm communities. NTHI variants within biofilms have on their surfaces lipooligosaccharides containing sialic acid (NeuAc) and phosphorylcholine (PCho). Our work showed that NeuAc promotes biofilm formation, but we observed no defect in the initial stages of biofilm formation for mutants lacking PCho. In this study, we asked if alterations in NTHI PCho content affect later stages of biofilm maturation. Biofilm communities were compared for NTHI 2019 and isogenic mutants that either lacked PCho (NTHI 2019 licD) or were constitutively locked in the PCho-positive phase (NTHI 2019 lic ON ). Transformants expressing green fluorescent protein were cultured in continuous-flow biofilms and analyzed by confocal laser scanning microscopy. COMSTAT was used to quantify different biofilm parameters. PCho expression correlated significantly with increased biofilm thickness, surface coverage, and total biomass, as well as with a decrease in biofilm roughness. Comparable results were obtained by scanning electron microscopy. Analysis of thin sections of biofilms by transmission electron microscopy revealed shedding of outer membrane vesicles by NTHI bacteria within biofilms and staining of matrix material with ruthenium red in biofilms formed by NTHI 2019 lic ON . The biofilms of all three strains were comparable in viability, the presence of extracellular DNA, and the presence of sialylated moieties on or between bacteria. In vivo infection studies using the chinchilla model of otitis media showed a direct correlation between PCho expression and biofilm formation within the middle-ear chamber and an inverse relationship between PCho and persistence in the planktonic phase in middle-ear effusions. Collectively, these data show that PCho correlates with, and may promote, the maturation of NTHI biofilms. Further, this structure may be disadvantageous in the planktonic phase.
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Su, Yu-Ching, Emma Mattsson, Birendra Singh, Farshid Jalalvand, Timothy F. Murphy, and Kristian Riesbeck. "The Laminin Interactome: A Multifactorial Laminin-Binding Strategy by Nontypeable Haemophilus influenzae for Effective Adherence and Colonization." Journal of Infectious Diseases 220, no. 6 (April 29, 2019): 1049–60. http://dx.doi.org/10.1093/infdis/jiz217.
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Abstract Laminin is a well-defined component of the airway basement membrane (BM). Efficient binding of laminin via multiple interactions is important for nontypeable Haemophilus influenzae (NTHi) colonization in the airway mucosa. In this study, we identified elongation factor thermo-unstable (EF-Tu), l-lactate dehydrogenase (LDH), protein D (PD), and peptidoglycan-associated lipoprotein P6 as novel laminin-binding proteins (Lbps) of NTHi. In parallel with other well-studied Lbps (protein 4 [P4], protein E [PE], protein F [PF], and Haemophilus adhesion and penetration protein [Hap]), EF-Tu, LDH, PD, and P6 exhibited interactions with laminin, and mediated NTHi laminin-dependent adherence to pulmonary epithelial cell lines. More importantly, the NTHi laminin interactome consisting of the well-studied and novel Lbps recognized laminin LG domains from the subunit α chains of laminin-111 and -332, the latter isoform of which is the main laminin in the airway BM. The NTHi interactome mainly targeted multiple heparin-binding domains of laminin. In conclusion, the NTHi interactome exhibited a high plasticity of interactions with different laminin isoforms via multiple heparin-binding sites.
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Li, Wenchao, Xinyun Zhang, Ying Yang, Qingqin Yin, Yan Wang, Yong Li, Chuan Wang, et al. "Recognition of conserved antigens by Th17 cells provides broad protection against pulmonaryHaemophilus influenzaeinfection." Proceedings of the National Academy of Sciences 115, no. 30 (July 9, 2018): E7149—E7157. http://dx.doi.org/10.1073/pnas.1802261115.
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NontypeableHaemophilus influenzae(NTHi) is a major cause of community acquired pneumonia and exacerbation of chronic obstructive pulmonary disease. A current effort in NTHi vaccine development has focused on generating humoral responses and has been greatly impeded by antigenic variation among the numerous circulating NTHi strains. In this study, we showed that pulmonary immunization of mice with killed NTHi generated broad protection against lung infection by different strains. While passive transfer of immune antibodies protected only against the homologous strain, transfer of immune T cells conferred protection against both homologous and heterologous strains. Further characterization revealed a strong Th17 response that was cross-reactive with different NTHi strains. Responding Th17 cells recognized both cytosolic and membrane-associated antigens, while immune antibodies preferentially responded to surface antigens and were highly strain specific. We further identified several conserved proteins recognized by lung Th17 cells during NTHi infection. Two proteins yielding the strongest responses were tested as vaccine candidates by immunization of mice with purified proteins plus an adjuvant. Immunization induced antigen-specific Th17 cells that recognized different strains and, upon adoptive transfer, conferred protection. Furthermore, immunized mice were protected against challenge with not only NTHi strains but also a fully virulent, encapsulated strain. Together, these results show that the immune mechanism of cross-protection against pneumonia involves Th17 cells, which respond to a broad spectrum of antigens, including those that are highly conserved among NTHi strains. These mechanistic insights suggest that inclusion of Th17 antigens in subunit vaccines offers the advantage of inducing broad protection and complements the current antibody-based approaches.
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Berenson, Charles S., Timothy F. Murphy, Catherine T. Wrona, and Sanjay Sethi. "Outer Membrane Protein P6 of Nontypeable Haemophilus influenzae Is a Potent and Selective Inducer of Human Macrophage Proinflammatory Cytokines." Infection and Immunity 73, no. 5 (May 2005): 2728–35. http://dx.doi.org/10.1128/iai.73.5.2728-2735.2005.
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ABSTRACT Interactions of nontypeable Haemophilus influenzae (NTHI) with human macrophages contribute to the pathogenesis of NTHI-induced infection in humans. However, the immunologic mechanisms that initiate and perpetuate NTHI-mediated macrophage responses have not been well explored. Outer membrane protein (OMP) P6 is a conserved lipoprotein expressed by NTHI in vivo that possesses a Pam3Cys terminal motif, characteristic of immunoactive bacterial lipoproteins associated with Toll-like receptor signaling. We theorized that OMP P6 is a potent immunomodulator of human macrophages. To test this hypothesis, we purified OMP P6 as well as OMP P2, the predominant NTHI outer membrane protein, and lipooligosaccharide (LOS), the specific endotoxin of NTHI, from NTHI strain 1479. Human blood monocyte-derived macrophages, purified from healthy donors, were incubated with each outer membrane constituent, and cytokine production of macrophage supernatants interleukin-1β (IL-1β), tumor necrosis factor α (TNF-α), IL-10, IL-12, and IL-8 was measured. OMP P6 selectively upregulated IL-10, TNF-α, and IL-8. While OMP P6 (0.1 μg/ml for 8 h) elicited slightly greater concentrations of IL-10, it resulted in over ninefold greater concentrations of TNF-α and over fourfold greater concentrations of IL-8 than did OMP P2. OMP P6 at doses as low as 10 pg/ml was still effective at induction of macrophage IL-8, while OMP P2 and LOS were not. OMP P6 of NTHI is a specific trigger of bacteria-induced human macrophage inflammatory events, with IL-8 and TNF-α as key effectors of P6-induced macrophage responses.
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Cevik, Muge, Olga L. Moncayo-Nieto, and Margaret J. Evans. "Non-typeable Haemophilus influenzae-associated early pregnancy loss: an emerging neonatal and maternal pathogen." Infection 48, no. 2 (September 23, 2019): 285–88. http://dx.doi.org/10.1007/s15010-019-01359-6.
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Abstract Objectives There is increasing evidence indicating an association between invasive non-typeable Haemophilus influenzae (NTHi) infection in pregnancy and early pregnancy loss. As the diagnosis relies on microbiological investigation of post-mortem placental and foetal samples, a significant proportion of NTHi-related pregnancy loss remains unrecognised. To better characterise NTHi in septic abortion, we report NTHi cases associated with early pregnancy loss. Methods We reviewed all post-mortems at <24 weeks gestation with histologically proven acute chorioamnionitis on placental histology and enrolled cases with at least one matched foetal and placental sample culture positive for NTHi. The study was approved by the NHS Lothian Caldicott Guardian. Results In our cohort, invasive NTHi has accounted for 20% of infections associated with early pregnancy loss prior to 24 weeks gestation. All patients were young and healthy pregnant women at < 20 weeks' gestation who presented with abdominal pain, PV bleed /discharge and were septic at the time of presentation. One patient with previous history of miscarriage who presented with cervical incompetence had more severe pathology suggestive of early intrauterine pneumonia. Conclusion The burden of invasive NTHi disease in early pregnancy loss is likely to be much larger than currently recognised. NTHi should be considered in pregnant women presenting with abdominal pain and PV bleed/discharge in whom clinical signs of sepsis are present. Active surveillance should be considered in this patient group including septic abortion to capture the true prevalence of this emerging pathogen to inform preventative and therapeutic approaches.
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Hutton, Andrew, Dr Karl Staples, Dr Tom Wilkinson, and Jane Warner. "Can human lung fibroblasts present antigen to autologous T cells? (MUC1P.911)." Journal of Immunology 194, no. 1_Supplement (May 1, 2015): 64.12. http://dx.doi.org/10.4049/jimmunol.194.supp.64.12.
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Abstract HLF are a major structural cell in the lung, and their ability to influence CD4 T cells in an Ag-specific manner is unknown. Recurrent bacterial infections, a hallmark of COPD, may involve bacterial presence in the mesenchyme and HLF-Tcell interaction. We have investigated the effect of non-typeable H.Influenzae (NTHi) on HLF immune synapse molecule expression and their ability to stimulate T cells. Lung tissue from patients undergoing resection surgery was collagenase digested and CD45-CD90+ HLF were demonstrated to express HLA-DR at baseline. HLF outgrown from this tissue were cultured and stimulated with IFNγ and/or NTHi for 48h. IFNγ upregulated both ICAM-1 5.6-fold (P<0.001, N=23) and HLA-DR 16.2-fold (P<0.001, N=20). Exposure to NTHi increased ICAM-1 6.1-fold (P<0.001, N=6) but not HLA-DR expression. HLF also expressed low levels of CD40 which increased 2.2-fold upon NTHi stimulation, but no detectable CD80 or 86 (N=5). To assess Ag presentation, HLFs were co-cultured with NTHi-specific autologous T cells overnight and cytokine production measured by FACS. Results indicate HLF preconditioned with IFNγ+NTHi could stimulate autologous T cells raised against NTHi Ags to produce IFNγ or IL-17A. Blocking experiments showed this stimulation to be reliant upon fibroblast HLA-DR expression. In conclusion, NTHi can cause HLF to present antigen to and activate autologous T cells. HLF-activated T cells may cause the lung damage associated with bacterial infections in COPD.
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Zagursky, Robert J., Peggy Ooi, Kevin F. Jones, Michael J. Fiske, Robert P. Smith, and Bruce A. Green. "Identification of a Haemophilus influenzae5′-Nucleotidase Protein: Cloning of the nucA Gene and Immunogenicity and Characterization of the NucA Protein." Infection and Immunity 68, no. 5 (May 1, 2000): 2525–34. http://dx.doi.org/10.1128/iai.68.5.2525-2534.2000.
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ABSTRACT We report on the identification of a surface-exposed, highly conserved, immunogenic nontypeable Haemophilus influenzae(NTHi) protein, which elicits cross-reactive bactericidal antibodies against NTHi. The protein was extracted from NTHi strain P860295 with KSCN and purified; it migrated as a single band on a sodium dodecyl sulfate-polyacrylamide gel with an apparent molecular mass of 63 kDa. Mouse antiserum generated against the purified protein was reactive on whole-cell enzyme-linked immunosorbent assay (ELISA) with seven NTHi strains and type b Eagan and Whittier strains and exhibited bactericidal activity to homologous and heterologous NTHi strains. However, the protein is made in small amounts in NTHi as corroborated by immunoelectron microscopy. To further study this protein, we cloned, sequenced, and expressed it recombinantly in Escherichia coli. The recombinant protein is localized in the periplasm ofE. coli and has been purified to homogeneity. Both the recombinant and native proteins possess 5′-nucleotidase activity; hence, the protein has been called NucA. Mouse antiserum directed against the recombinant NucA protein was reactive on Western immunoblots and whole-cell ELISA with all H. influenzaestrains tested including Eagan and was bactericidal for two heterologous strains tested. The antiserum also resulted in a log reduction in bacteremia, in an infant-rat protection study withH. influenzae type b as the challenge strain. These features suggest that NucA is a potential subunit vaccine candidate against NTHi disease.
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Wang, Beinan, P. Patrick Cleary, Haidong Xu, and Jian-Dong Li. "Up-Regulation of Interleukin-8 by Novel Small Cytoplasmic Molecules of Nontypeable Haemophilus influenzae via p38 and Extracellular Signal-Regulated Kinase Pathways." Infection and Immunity 71, no. 10 (October 2003): 5523–30. http://dx.doi.org/10.1128/iai.71.10.5523-5530.2003.
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ABSTRACT Nontypeable Haemophilus influenzae (NTHI) is an important etiological agent of otitis media (OM) and of exacerbated chronic obstructive pulmonary diseases (COPD). Inflammation is a hallmark of both diseases. Interleukin-8 (IL-8), one of the important inflammatory mediators, is induced by NTHI and may play a significant role in the pathogenesis of these diseases. Our studies demonstrated that a soluble cytoplasmic fraction (SCF) from NTHI induced much greater IL-8 expression by human epithelial cells than did NTHI lipooligosaccharides and envelope proteins. The IL-8-inducing activity was associated with molecules of ≤3 kDa from SCF and was peptidase and lipase sensitive, suggesting that small lipopeptides are responsible for the strong IL-8 induction. Moreover, multiple intracellular signaling pathways were activated in response to cytoplasmic molecules. The results indicated that the p38 mitogen-activated protein kinase (MAPK) and Src-dependent Raf-1-Mek1/2-extracellular signal-regulated kinase mitogen-activated protein kinase (ERK MAPK) pathways are required for NTHI-induced IL-8 production. In contrast, the phosphatidylinositol 3-kinase (PI3K)-Akt pathway did not affect IL-8 expression, although this pathway was concomitantly activated upon exposure to NTHI SCF. The PI3K-Akt pathway was also directly activated by IL-8 and significantly inhibited by an antagonist of IL-8 receptors during NTHI stimulation. These results indicated that the PI3K-Akt pathway is activated in response to IL-8 that is induced by NTHI and may lead to other important epithelial cell responses. This work provides insight into essential molecular and cellular events that may impact on the pathogenesis of OM and COPD and identifies rational targets for anti-inflammatory intervention.
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Pang, Bing, Dana Winn, Ryan Johnson, Wenzhou Hong, Shayla West-Barnette, Nancy Kock, and W. Edward Swords. "Lipooligosaccharides Containing Phosphorylcholine Delay Pulmonary Clearance of Nontypeable Haemophilus influenzae." Infection and Immunity 76, no. 5 (March 17, 2008): 2037–43. http://dx.doi.org/10.1128/iai.01716-07.
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ABSTRACT Nontypeable Haemophilus influenzae (NTHi) causes pulmonary infections in patients with chronic obstructive pulmonary disease and other mucociliary clearance defects. Like many bacteria inhabiting mucosal surfaces, NTHi produces lipooligosaccharide (LOS) endotoxins that lack the O side chain. Persistent NTHi populations express a discrete subset of LOS glycoforms, including those containing phosphorylcholine (PCho). In this study, we compared two NTHi strains with isogenic mutants lacking PCho for clearance from mice following pulmonary infection. Consistent with data from other model systems, populations of the strains NTHi 2019 and NTHi 86-028NP recovered from mouse lung contained an increased proportion of PCho+ variants compared to that in the inocula. PCho− mutants were more rapidly cleared. Serial passage of NTHi increased both PCho content and bacterial resistance to clearance, and no such increases were observed for PCho− mutants. Increased PCho content was also observed in NTHi populations within non-endotoxin-responsive C3H/HeJ and Toll-like receptor 4 null (TLR4−/−) mice, albeit at later times postinfection. Changes in bacterial subpopulations and clearance were unaffected in TLR2−/− mice compared to the subpopulations in and clearance from mice of the parental strain. The clearance of PCho− mutants occurred at earlier time points in both strain backgrounds and in all types of mice. Comparison of bacterial populations in lung tissue cryosections by immunofluorescent staining showed sparse bacteria within the air spaces of C57BL/6 mice and large bacterial aggregates within the lungs of MyD88−/− mice. These results indicate that PCho promotes bacterial resistance to pulmonary clearance early in infection in a manner that is at least partially independent of the TLR4 pathway.
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Atto, Brianna, Dale Kunde, David A. Gell, and Stephen Tristram. "Haemophilin-Producing Strains of Haemophilus haemolyticus Protect Respiratory Epithelia from NTHi Colonisation and Internalisation." Pathogens 10, no. 1 (January 1, 2021): 29. http://dx.doi.org/10.3390/pathogens10010029.
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Nontypeable Haemophilus influenzae (NTHi) is a significant respiratory tract pathogen responsible for infections that collectively pose a substantial health and socioeconomic burden. The clinical course of these infections is largely dictated by NTHi interactions with host respiratory epithelia, and thus, approaches that disrupt colonisation and invasion may have significant therapeutic potential. Survival, successful host–cell interactions, and pathogenesis are reliant on NTHi’s ability to sequester host-derived haem. Previously, we demonstrated the therapeutic potential of exploiting this haem-dependence using a closely related competitor bacterium, Haemophilus haemolyticus (Hh). Hh strains capable of producing the novel haem-binding protein haemophilin (Hpl) possessed potent inhibitory activity by restricting NTHi access to haem in a broth co-culture environment. Here, we extend this work to cell culture models that more closely represent the human respiratory epithelium and show that Hh strains with high levels of hpl expression protect epithelial cell line monolayers against adhesion and invasion by NTHi. Inhibitory activity was dependent on the level of Hpl production, which was stimulated by NTHi challenge and nasopharyngeal cell exposure. Provided these protective benefits translate to in vivo applications, Hpl-producing Hh may have probiotic utility against NTHi infections by inhibiting requisite nasopharyngeal colonisation.
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Harrison, Alistair, William C. Ray, Beth D. Baker, David W. Armbruster, Lauren O. Bakaletz, and Robert S. Munson. "The OxyR Regulon in Nontypeable Haemophilus influenzae." Journal of Bacteriology 189, no. 3 (December 1, 2006): 1004–12. http://dx.doi.org/10.1128/jb.01040-06.
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ABSTRACT Nontypeable Haemophilus influenzae (NTHi) is a gram-negative bacterium and a common commensal organism of the upper respiratory tract in humans. NTHi causes a number of diseases, including otitis media, sinusitis, conjunctivitis, exacerbations of chronic obstructive pulmonary disease, and bronchitis. During the course of colonization and infection, NTHi must withstand oxidative stress generated by insult due to multiple reactive oxygen species produced endogenously by other copathogens and by host cells. Using an NTHi-specific microarray containing oligonucleotides representing the 1821 open reading frames of the recently sequenced NTHi isolate 86-028NP, we have identified 40 genes in strain 86-028NP that are upregulated after induction of oxidative stress due to hydrogen peroxide. Further comparisons between the parent and an isogenic oxyR mutant identified a subset of 11 genes that were transcriptionally regulated by OxyR, a global regulator of oxidative stress. Interestingly, hydrogen peroxide induced the OxyR-independent upregulation of expression of the genes encoding components of multiple iron utilization systems. This finding suggested that careful balancing of levels of intracellular iron was important for minimizing the effects of oxidative stress during NTHi colonization and infection and that there are additional regulatory pathways involved in iron utilization.
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Bakaletz, Lauren O., and Laura A. Novotny. "Nontypeable Haemophilus influenzae (NTHi)." Trends in Microbiology 26, no. 8 (August 2018): 727–28. http://dx.doi.org/10.1016/j.tim.2018.05.001.
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Greiner, L. L., H. Watanabe, N. J. Phillips, J. Shao, A. Morgan, A. Zaleski, B. W. Gibson, and M. A. Apicella. "Nontypeable Haemophilus influenzae Strain 2019 Produces a Biofilm Containing N-Acetylneuraminic Acid That May Mimic Sialylated O-Linked Glycans." Infection and Immunity 72, no. 7 (July 2004): 4249–60. http://dx.doi.org/10.1128/iai.72.7.4249-4260.2004.
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ABSTRACT Previous studies suggested that nontypeable Haemophilus influenzae (NTHI) can form biofilms during human and chinchilla middle ear infections. Microscopic analysis of a 5-day biofilm of NTHI strain 2019 grown in a continuous-flow chamber revealed that the biofilm had a diffuse matrix interlaced with multiple water channels. Our studies showed that biofilm production was significantly decreased when a chemically defined medium lacking N-acetylneuraminic acid (sialic acid) was used. Based on these observations, we examined mutations in seven NTHI strain 2019 genes involved in carbohydrate and lipooligosaccharide biosynthesis. NTHI strain 2019 with mutations in the genes encoding CMP-N-acetylneuraminic acid synthetase (siaB), one of the three NTHI sialyltransferases (siaA), and the undecaprenyl-phosphate α-N-acetylglucosaminyltransferase homolog (wecA) produced significantly smaller amounts of biofilm. NTHI strain 2019 with mutations in genes encoding phosphoglucomutase (pgm), UDP-galactose-4-epimerase, and two other NTHI sialyltransferases (lic3A and lsgB) produced biofilms that were equivalent to or larger than the biofilms produced by the parent strain. The biofilm formed by the NTHI strain 2019pgm mutant was studied with Maackia amurensis fluorescein isothiocyanate (FITC)-conjugated and Sambucus nigra tetramethyl rhodamine isocyanate (TRITC)-conjugated lectins. S. nigra TRITC-conjugated lectin bound to this biofilm, while M. amurensis FITC-conjugated lectin did not. S. nigra TRITC-conjugated lectin binding was inhibited by incubation with α2,6-neuraminyllactose and by pretreatment of the biofilm with Vibrio cholerae neuraminidase. Matrix-assisted laser desorption ionization—time of flight mass spectometry analysis of lipooligosaccharides isolated from a biofilm, the planktonic phase, and plate-grown organisms showed that the levels of most sialylated glycoforms were two- to fourfold greater when the lipooligosaccharide was derived from planktonic or biofilm organisms. Our data indicate that NTHI strain 2019 produces a biofilm containing α2,6-linked sialic acid and that the sialic acid content of the lipooligosaccharides increases concomitant with the transition of organisms to a biofilm form.
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Brown, Mary Ashley, Maisha Jabeen, Gurpreet Bharj, and Timothy S. C. Hinks. "Non-typeable Haemophilus influenzae airways infection: the next treatable trait in asthma?" European Respiratory Review 31, no. 165 (September 20, 2022): 220008. http://dx.doi.org/10.1183/16000617.0008-2022.
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Asthma is a complex, heterogeneous condition that affects over 350 million people globally. It is characterised by bronchial hyperreactivity and airways inflammation. A subset display marked airway neutrophilia, associated with worse lung function, higher morbidity and poor response to treatment. In these individuals, recent metagenomic studies have identified persistent bacterial infection, particularly with non-encapsulated strains of the Gram-negative bacterium Haemophilus influenzae. Here we review knowledge of non-typeable H. influenzae (NTHi) in the microbiology of asthma, the immune consequences of mucosal NTHi infection, various immune evasion mechanisms, and the clinical implications of NTHi infection for phenotyping and targeted therapies in neutrophilic asthma. Airway neutrophilia is associated with production of neutrophil chemokines and proinflammatory cytokines in the airways, including interleukin (IL)-1β, IL-6, IL-8, IL-12, IL-17A and tumour necrosis factor. NTHi adheres to and invades the lower respiratory tract epithelium, inducing the NLR family pyrin domain containing 3 (NLRP3) and absent in melanoma 2 (AIM2) inflammasomes. NTHi reduces expression of tight-junction proteins, impairing epithelial integrity, and can persist intracellularly. NTHi interacts with rhinoviruses synergistically via upregulation of intracellular cell adhesion molecule 1 and promotion of a neutrophilic environment, to which NTHi is adapted. We highlight the clinical relevance of this emerging pathogen and its relevance for the efficacy of long-term macrolide therapy in airways diseases, we identify important unanswered questions and we propose future directions for research.
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Yang, Yan-ping, Sheena M. Loosmore, Brian J. Underdown, and Michel H. Klein. "Nasopharyngeal Colonization with Nontypeable Haemophilus influenzae in Chinchillas." Infection and Immunity 66, no. 5 (May 1, 1998): 1973–80. http://dx.doi.org/10.1128/iai.66.5.1973-1980.1998.
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ABSTRACT Colonization of the nasopharynx by a middle ear pathogen is the first step in the development of otitis media in humans. The establishment of an animal model of nasopharyngeal colonization would therefore be of great utility in assessing the potential protective ability of candidate vaccine antigens (especially adhesins) against otitis media. A chinchilla nasopharyngeal colonization model for nontypeable Haemophilus influenzae (NTHI) was developed with antibiotic-resistant strains. This model does not require coinfection with a virus. There was no significant difference in the efficiency of NTHI colonization between adult (1- to 2-year-old) and young (2- to 3-month-old) animals. However, the incidence of middle ear infection following nasopharyngeal colonization was significantly higher in young animals (83 to 89%) than in adult chinchillas (10 to 30%). Chinchillas that had recovered either from a previous middle ear infection caused by NTHI or from an infection by intranasal inoculation with NTHI were completely protected against nasopharyngeal colonization with a homologous strain and were found to be the best positive controls in protection studies. Systemic immunization of chinchillas with inactivated whole-cell preparations significantly protected animals not only against homologous NTHI colonization but also partially against heterologous NTHI infection. In all protected animals, significant serum anti-P6 and anti-HMW antibody responses were observed. The outer membrane P6 and high-molecular-weight (HMW) proteins appear to be promising candidate vaccine antigens to prevent nasopharyngeal colonization and middle ear infection caused by NTHI.
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Stull, Terrence, Paul Whitby, Daniel Morton, Huda Mussa, and Lucia Mirea. "15. A Novel Approach to Bacterial Vaccines: Haemophilus influenzae as a Paradigm." Open Forum Infectious Diseases 7, Supplement_1 (October 1, 2020): S30. http://dx.doi.org/10.1093/ofid/ofaa439.060.
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Abstract Background The H. influenzae type b vaccines target the type b capsule and therefore have no impact on the nontypable (unencapsulated) H. influenzae (NTHi). NTHi has become the most common cause of otitis media and is the most common isolate from patients with exacerbations of Chronic Obstructive Pulmonary Disease (COPD). Therefore, NTHi is an appropriate target for vaccine development. Methods To characterize potential vaccine targets, the core outer proteins of NTHi present in the available sequenced genomes were identified through genomic bioinformatics. The structures of the outer proteins were analyzed through comparison with the available structures of homologues characterized by X-ray crystallography. Sequenced conserved outer regions of these proteins were analyzed for their protective capacity in the infant rat model of H. influenzae infection. Results Nine peptides that were protective in the infant rat model were used in a novel vaccine to immunize chinchillas, the most established animal model of otitis media. Chinchillas (40 vaccinated and 41 controls) were infected with NTHi 86-028NP. The vaccinated group cleared infection more quickly than the control group as indicated by significantly decreased positive findings on video-otoscopy (p< 0.0001) and tympanometry (p=0.0002) on day 7, and presence of middle ear fluid obtained by aspiration (p=0.0001) on day 10 post infection. Similarly, in the mouse model of NTHi pulmonary clearance, the vaccinated group (n=5) reduced infection more rapidly than the control group (n= 5), p=0.008. Conclusion These data demonstrate the effectiveness of the Bacterial Vaccine Polypeptide methodology in development of a vaccine against NTHi with protection in relevant preclinical models of both otitis media and pulmonary clearance. The methods are applicable to other bacteria, and this approach to a Bacterial Vaccine Polypeptide against NTHi serves as a paradigm for development of similar vaccines to protect against other bacterial infections. Disclosures All Authors: No reported disclosures