| 1. |
- Jalalvand, Farshid, et al.
(författare)
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Haemophilus influenzae Protein F Mediates Binding to Laminin and Human Pulmonary Epithelial Cells.
- 2012
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Ingår i: Journal of Infectious Diseases. - : Oxford University Press (OUP). - 1537-6613 .- 0022-1899. ; 207:5, s. 803-813
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Tidskriftsartikel (refereegranskat)abstract
- The mucosal pathogen non-typeable Haemophilus influenzae (NTHi) adheres to the respiratory epithelium, or in the case of epithelial damage, to the underlying basement membrane and extracellular matrix that amongst other proteins consists of laminin. We have recently identified Protein F, an ABC-transporter involved in NTHi immune evasion. Homology modeling of the Protein F tertiary structure revealed a strong resemblance to the streptococcal laminin-binding proteins Lbp and Lmb. Here, we show that Protein F promotes binding of NTHi to laminin and primary bronchial epithelial cells. Analyses with recombinant proteins and synthetic peptides revealed that the N-terminal part of Protein F contains the host-interacting region. Moreover, Protein F exists in all clinical isolates, and isogenic NTHi Δhpf mutants display significantly reduced binding to laminin and epithelial cells. We thus suggest Protein F as an important and ubiquitous NTHi adhesin.
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| 2. |
- Jalalvand, Farshid, et al.
(författare)
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Haemophilus influenzae: recent advances in the understanding of molecular pathogenesis and polymicrobial infections.
- 2014
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Ingår i: Current Opinion in Infectious Diseases. - 1473-6527. ; 27:3, s. 268-274
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Forskningsöversikt (refereegranskat)abstract
- Non-typeable Haemophilus influenzae (NTHi) is a human-specific mucosal pathogen and one of the most common causes of bacterial infections in children and patients with chronic obstructive pulmonary disease. It is also frequently found in polymicrobial superinfections. Great strides have recently been made in the understanding of the molecular mechanisms underlying NTHi pathogenesis.
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| 3. |
- Jalalvand, Farshid, et al.
(författare)
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Impact of immunization with Protein F on pulmonary clearance of nontypeable Haemophilus influenzae.
- 2014
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Ingår i: Vaccine. - : Elsevier BV. - 1873-2518 .- 0264-410X. ; 32:20, s. 2261-2264
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Tidskriftsartikel (refereegranskat)abstract
- Nontypeable Haemophilus influenzae (NTHi) is one of the main aetiologies of childhood bacterial infections as well as exacerbations in COPD patients. Currently, no licensed NTHi vaccine exists. In the present study, we evaluated the potential of the conserved and ubiquitous surface protein Haemophilus Protein F (PF) as a vaccine candidate. Our results show that incubation of NTHi with anti-PF antibodies significantly increased the opsonophygocytosis of human promyelocytic leukemia cell line-derived granulocytes, leading to efficient killing of the bacteria (P≤0.05). The presence of anti-PF IgG titers in healthy adults (n=60) was investigated, and we found that 26% of healthy blood donors carried antibodies with the main antigenic epitope being PF(23-48). Finally, mice immunized with PF(23-48) attained a significantly increased capacity to clear NTHi as compared to a control group immunized with a peptide derived from Moraxella catarrhalis β-lactamase (P≤0.05). Taken together, our results indicate that PF is a potential NTHi-vaccine candidate.
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| 4. |
- Jalalvand, Farshid, et al.
(författare)
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Protein domain-dependent vesiculation of Lipoprotein A, a protein that is important in cell wall synthesis and fitness of the human respiratory pathogen Haemophilus influenzae
- 2022
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Ingår i: Frontiers in cellular and infection microbiology. - : Frontiers Media SA. - 2235-2988. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- The human pathogen Haemophilus influenzae causes respiratory tract infections and is commonly associated with prolonged carriage in patients with chronic obstructive pulmonary disease. Production of outer membrane vesicles (OMVs) is a ubiquitous phenomenon observed in Gram-negative bacteria including H. influenzae. OMVs play an important role in various interactions with the human host; from neutralization of antibodies and complement activation to spread of antimicrobial resistance. Upon vesiculation certain proteins are found in OMVs and some proteins are retained at the cell membrane. The mechanism for this phenomenon is not fully elucidated. We employed mass spectrometry to study vesiculation and the fate of proteins in the outer membrane. Functional groups of proteins were differentially distributed on the cell surface and in OMVs. Despite its supposedly periplasmic and outer membrane location, we found that the peptidoglycan synthase-activator Lipoprotein A (LpoA) was accumulated in OMVs relative to membrane fractions. A mutant devoid of LpoA lost its fitness as revealed by growth and electron microscopy. Furthermore, high-pressure liquid chromatography disclosed a lower concentration (55%) of peptidoglycan in the LpoA-deficient H. influenzae compared to the parent wild type bacterium. Using an LpoA-mNeonGreen fusion protein and fluorescence microscopy, we observed that LpoA was enriched in “foci” in the cell envelope, and further located in the septum during cell division. To define the fate of LpoA, C-terminally truncated LpoA-variants were constructed, and we found that the LpoA C-terminal domain promoted optimal transportation to the OMVs as revealed by flow cytometry. Taken together, our study highlights the importance of LpoA for H. influenzae peptidoglycan biogenesis and provides novel insights into cell wall integrity and OMV production.
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| 5. |
- Jalalvand, Farshid
(författare)
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The Colonization Strategies of Nontypeable Haemophilus influenzae - Bacterial Colonization Factors and Vaccine Development
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Nontypeable Haemophilus influenzae (NTHi) is a remarkably proficient colonizer of the human respiratory tract. The aim of this thesis has been to characterize currently known, and identify novel, bacterial factors involved in the key processes of colonization and pathogenesis, namely adherence to host tissue and evasion of the host innate immunity. We have shown that the NTHi virulence factor Protein E mediates innate immune evasion by interacting with the C-terminus of host complement down-regulatory protein vitronectin. This interaction delayed the formation of the membrane attack complex on the bacterial surface and increased the bacterial serum-resistance. Furthermore, we identified a novel surface-exposed virulence factor designated Haemophilus Protein F that also sequesters vitronectin on the surface of the bacteria, thus rendering the pathogen more serum-resistant. Moreover, we have revealed that Protein F mediates bacterial adherence to primary human bronchial cells and to the extracellular matrix protein laminin. Isogenic mutants devoid of Protein F were observed to adhere significantly less to vitronectin, epithelial cells and laminin. These interactions were characterized at the molecular level, and the N-terminus of Protein F was shown to contain the host-interacting region. Finally, we showed that immunization with Protein F conferred increased pulmonary clearance of NTHi in vivo. In summary, we have identified, and at the molecular level characterized, novel virulence factors that may be suitable for future vaccine development.
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| 6. |
- Jalalvand, Farshid, et al.
(författare)
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Update on non-typeable Haemophilus influenzae-mediated disease and vaccine development
- 2018
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Ingår i: Expert Review of Vaccines. - : Informa UK Limited. - 1476-0584 .- 1744-8395. ; 17:6, s. 503-512
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Forskningsöversikt (refereegranskat)abstract
- Introduction: Non-typeable Haemophilus influenzae (NTHi) has attracted more interest in recent years due to an increased prevalence of infections caused by the pathogen. This upsurge is at least partly ascribed to the introduction of the pneumococcal conjugated vaccines that has resulted in an aetiological shift in NTHi’s favor with respect to upper respiratory tract infections. Moreover, an increased antimicrobial resistance has been associated with the pathogen, a fact that further strengthens the case for novel vaccine development. Areas covered: A background to NTHi-mediated diseases and pathogenesis is outlined. The literature in the field of NTHi vaccine antigens and clinical trials is reviewed with focus on data added to scientific databases in the last two years. Various vaccine development strategies are conceptually discussed. Expert commentary: Several promising vaccine antigens have been defined in recent years. A multicomponent protein-based vaccine, potentially boosted with extracellular vesicles, would constitute a suitable path going forward. Of note, however, a clinical trial investigating the efficacy of a combined NTHi/Moraxella catarrhalis vaccine to prevent infections in chronic obstructive pulmonary disease (COPD) patients has been initiated. But, as this clinical trial has not yet concluded, and its results are thus unknown, investigations of NTHi pathogenesis must determinedly continue.
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| 7. |
- Singh, Birendra, et al.
(författare)
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Haemophilus influenzae protein E recognizes the C-terminal domain of vitronectin and modulates the membrane attack complex.
- 2011
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Ingår i: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 81, s. 80-98
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Tidskriftsartikel (refereegranskat)abstract
- Haemophilus influenzae protein E (PE) is a 16 kDa adhesin that induces a pro-inflammatory immune response in lung epithelial cells. The active epithelial binding region comprising amino acids PE 84-108 also interferes with complement-mediated bacterial killing by capturing vitronectin (Vn) that prevents complement deposition and formation of the membrane attack complex (MAC). Here, the interaction between PE and Vn was characterized using site-directed mutagenesis. Protein E variants were produced both in soluble forms and in surface-expressed molecules on Escherichia coli. Mutations within PE(84-108) in the full-length molecule revealed that K85 and R86 residues were important for the Vn binding. Bactericidal activity against H. influenzae was higher in human serum pre-treated with full-length PE as compared with serum incubated with PE(K85E, R86D) , suggesting that PE quenched Vn. A series of truncated Vn molecules revealed that the C-terminal domain comprising Vn(353-363) harboured the major binding region for PE. Interestingly, MAC deposition was significantly higher on mutants devoid of PE due to a decreased Vn-binding capacity when compared with wild-type H. influenzae. Our results define a fine-tuned interaction between H. influenzae and the innate immune system, and identify the mode of control of the MAC that is important for pathogen complement evasion.
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| 8. |
- Singh, Birendra, et al.
(författare)
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Human pathogens utilize host extracellular matrix proteins laminin and collagen for adhesion and invasion of the host.
- 2012
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Ingår i: FEMS Microbiology Reviews. - : Oxford University Press (OUP). - 1574-6976. ; 36:6, s. 1122-1180
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Tidskriftsartikel (refereegranskat)abstract
- Laminin (Ln) and collagen are multifunctional glycoproteins that play an important role in cellular morphogenesis, cell signaling, tissue repair and cell migration. These proteins are ubiquitously present in tissues as a part of the basement membrane (BM), constitute a protective layer around blood capillaries, and are included in the extracellular matrix (ECM). As a component of BMs, both Lns and collagen(s) are found in the blood-brain barrier, and thus function as major mechanical containment molecules that protect tissues from pathogens. Invasive pathogens breach the basal lamina and degrade ECM proteins of interstitial spaces and connective tissues by using various ECM degrading proteases or surface-bound plasminogen and matrix metalloproteinases recruited from the host. Most pathogens associated with the respiratory, gastrointestinal, or urogenital tracts, as well as the CNS or the skin have the capacity to bind and degrade Lns and collagen(s) in order to adhere to and invade host tissues. In this review we focus on the adaptability of various pathogens to utilize these ECM proteins as enhancers for adhesion to host tissues or as a targets for degradation in order to breach the cellular barriers. The major pathogens discussed are Streptococcus, Staphylococcus, Pseudomonas, Salmonella, Yersinia, Treponema, Mycobacterium, Clostridium, Listeria, Porphyromonas and Haemophilus; Candida, Aspergillus, Pneumocystis, Cryptococcus and Coccidioides; Acanthamoeba, Trypanosoma and Trichomonas; Retrovirus and papilloma virus © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.
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| 9. |
- Su, Shanice Yc, et al.
(författare)
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The Laminin Interactome : A Multifactorial Laminin-Binding Strategy by Nontypeable Haemophilus influenzae for Effective Adherence and Colonization
- 2019
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Ingår i: The Journal of infectious diseases. - : Oxford University Press (OUP). - 1537-6613 .- 0022-1899. ; 220:6, s. 1049-1060
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Tidskriftsartikel (refereegranskat)abstract
- Laminin is a well-defined component of the airway basement membrane (BM). Efficient binding of laminin via multiple interactions is important for nontypeable Haemophilusinfluenzae (NTHi) colonization in the airway mucosa. Here we identified elongation factor thermo-unstable (EF-Tu), L-lactate dehydrogenase (LDH), Protein D and peptidoglycan-associated lipoprotein P6 as novel laminin-binding proteins (Lbps) of NTHi. In parallel with other well-studied Lbps (P4, PE, PF and Hap), EF-Tu, LDH, PD and P6 exhibited interactions with laminin, and mediated NTHi laminin-dependent adherence to pulmonary epithelial cell lines. 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, of which the latter isoform 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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| 10. |
- Su, Yu-Ching, et al.
(författare)
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Haemophilus influenzae acquires vitronectin via the ubiquitous Protein F to subvert host innate immunity.
- 2013
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Ingår i: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 87:6, s. 1245-1266
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Tidskriftsartikel (refereegranskat)abstract
- Acquisition of the complement inhibitor vitronectin (Vn) is important for the respiratory tract pathogen nontypeable Haemophilus influenzae (NTHi) to escape complement-mediated killing. NTHi actively recruits Vn, and we previously showed that this interaction involves Protein E (PE). Here we describe a second Vn-binding protein, a 30 kDa Yersinia YfeA homologue designated as Protein F (PF). An isogenic NTHi 3655Δhpf mutant devoid of PF displayed a reduced binding of Vn, and was consequently more sensitive to killing by human serum compared with the wild type. Surface expression of PF on Escherichia coli conferred binding of Vn that resulted in a serum resistant phenotype. Molecular analyses revealed that the N-terminal of PF (Lys23-Glu48) bound to the C-terminal of Vn (Phe352-Ser374) without disrupting the inhibitory role of Vn on the membrane attack complex. The PF-Vn complex actively delayed C9 deposition on PF-expressing bacteria. Comparative studies of binding affinity and multiple mutants demonstrated that both PE and PF contribute individually to NTHi serum survival. PF was highly conserved and ubiquitously expressed in a series of randomly selected NTHi clinical isolates (n = 18). In conclusion, the multifaceted binding of Vn is beneficial for NTHi survival in serum and may contribute to successful colonization and consequently infection.
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| 11. |
- Su, Yu Ching, et al.
(författare)
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Haemophilus Protein F Orthologs of Pathogens Infecting the Airways : Exploiting Host Laminin at Heparin-Binding Sites for Maximal Adherence to Epithelial Cells
- 2017
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Ingår i: The Journal of infectious diseases. - : Oxford University Press (OUP). - 1537-6613 .- 0022-1899. ; 216:10, s. 1303-1307
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Tidskriftsartikel (refereegranskat)abstract
- Haemophilus influenzae protein F (PF) is an important virulence factor interacting with laminin, an extracellular matrix protein ubiquitously expressed in the respiratory tract. Here we defined PF orthologs in Pseudomonas aeruginosa, Moraxella catarrhalis, and Staphylococcus aureus, bacteria that occasionally colonize and infect the human airways. Despite low sequence homology (48.2%-77.3% similarity), all orthologs (Paf, AfeA, and MntC) interacted with laminin. Interestingly, all proteins bound at the heparin-binding sites of laminin, including the globular domains, and also attached to laminin expressed on respiratory epithelial cells. Laminin is thus a highly important target for PF orthologs of the bacterial species examined.
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| 12. |
- Su, Yu Ching, et al.
(författare)
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Non-typeable Haemophilus influenzae major outer membrane protein P5 contributes to bacterial membrane stability, and affects the membrane protein composition crucial for interactions with the human host
- 2023
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Ingår i: Frontiers in Cellular and Infection Microbiology. - : Frontiers Media S.A.. - 2235-2988. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- Non-typeable Haemophilus influenzae (NTHi) is a Gram-negative human pathogen that causes a wide range of airway diseases. NTHi has a plethora of mechanisms to colonize while evading the host immune system for the establishment of infection. We previously showed that the outer membrane protein P5 contributes to bacterial serum resistance by the recruitment of complement regulators. Here, we report a novel role of P5 in maintaining bacterial outer membrane (OM) integrity and protein composition important for NTHi-host interactions. In silico analysis revealed a peptidoglycan-binding motif at the periplasmic C-terminal domain (CTD) of P5. In a peptidoglycan-binding assay, the CTD of P5 (P5CTD) formed a complex with peptidoglycan. Protein profiling analysis revealed that deletion of CTD or the entire P5 changed the membrane protein composition of the strains NTHi 3655Δp5CTD and NTHi 3655Δp5, respectively. Relative abundance of several membrane-associated virulence factors that are crucial for adherence to the airway mucosa, and serum resistance were altered. This was also supported by similar attenuated pathogenic phenotypes observed in both NTHi 3655Δp5CTD and NTHi 3655Δp5. We found (i) a decreased adherence to airway epithelial cells and fibronectin, (ii) increased complement-mediated killing, and (iii) increased sensitivity to the β-lactam antibiotics in both mutants compared to NTHi 3655 wild-type. These mutants were also more sensitive to lysis at hyperosmotic conditions and hypervesiculated compared to the parent wild-type bacteria. In conclusion, our results suggest that P5 is important for bacterial OM stability, which ultimately affects the membrane proteome and NTHi pathogenesis.
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| 13. |
- Su, Yu Ching, et al.
(författare)
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The interplay between immune response and bacterial infection in COPD : Focus Upon non-typeable Haemophilus influenzae
- 2018
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Ingår i: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 9:NOV
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Forskningsöversikt (refereegranskat)abstract
- Chronic obstructive pulmonary disease (COPD) is a debilitating respiratory disease and one of the leading causes of morbidity and mortality worldwide. It is characterized by persistent respiratory symptoms and airflow limitation due to abnormalities in the lower airway following consistent exposure to noxious particles or gases. Acute exacerbations of COPD (AECOPD) are characterized by increased cough, purulent sputum production, and dyspnea. The AECOPD is mostly associated with infection caused by common cold viruses or bacteria, or co-infections. Chronic and persistent infection by nontypeable Haemophilus influenzae (NTHi), a Gram-negative coccobacillus, contributes to almost half of the infective exacerbations caused by bacteria. This is supported by reports that NTHi is commonly isolated in the sputum from COPD patients during exacerbations. Persistent colonization of NTHi in the lower airway requires a plethora of phenotypic adaptation and virulent mechanisms that are developed over time to cope with changing environmental pressures in the airway such as host immuno-inflammatory response. Chronic inhalation of noxious irritants in COPD causes a changed balance in the lung microbiome, abnormal inflammatory response, and an impaired airway immune system. These conditions significantly provide an opportunistic platform for NTHi colonization and infection resulting in a “vicious circle.” Episodes of large inflammation as the consequences of multiple interactions between airway immune cells and NTHi, accumulatively contribute to COPD exacerbations and may result in worsening of the clinical status. In this review, we discuss in detail the interplay and crosstalk between airway immune residents and NTHi, and their effect in AECOPD for better understanding of NTHi pathogenesis in COPD patients.
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| 14. |
- Thofte, Oskar, et al.
(författare)
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EF-Tu From Non-typeable Haemophilus influenzae Is an Immunogenic Surface-Exposed Protein Targeted by Bactericidal Antibodies
- 2018
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Ingår i: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 9, s. 2910-2910
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Tidskriftsartikel (refereegranskat)abstract
- Non-typeable Haemophilus influenzae (NTHi), a commensal organism in pre-school children, is an opportunistic pathogen causing respiratory tract infections including acute otitis media. Adults suffering from chronic obstructive pulmonary disease (COPD) are persistently colonized by NTHi. Previous research has suggested that, in some bacterial species, the intracellular elongation factor thermo-unstable (EF-Tu) can moonlight as a surface protein upon host encounter. The aim of this study was to determine whether EF-Tu localizes to the surface of H. influenzae, and if such surface-associated EF-Tu is a target for bactericidal antibodies. Using flow cytometry, transmission immunoelectron microscopy, and epitope mapping, we demonstrated that EF-Tu is exposed at the surface of NTHi, and identified immunodominant epitopes of this protein. Rabbits immunized with whole-cell NTHi produced significantly more immunoglobulin G (IgG) directed against EF-Tu than against the NTHi outer membrane proteins D and F as revealed by enzyme-linked immunosorbent assays. Chemical cleavage of NTHi EF-Tu by cyanogen bromide (CNBr) followed by immunoblotting showed that the immunodominant epitopes were located within the central and C-terminal regions of the protein. Peptide epitope mapping by dot blot analysis further revealed four different immunodominant peptide sequences; EF-Tu41-65, EF-Tu161-185, EF-Tu221-245, and EF-Tu281-305. These epitopes were confirmed to be surface-exposed and accessible by peptide-specific antibodies in flow cytometry. We also analyzed whether antibodies raised against NTHi EF-Tu cross-react with other respiratory tract pathogens. Anti-EF-Tu IgG significantly detected EF-Tu on unencapsulated bacteria, including the Gram-negative H. parainfluenzae, H. haemolyticus, Moraxella catarrhalis and various Gram-positive Streptococci of the oral microbiome. In contrast, considerably less EF-Tu was observed at the surface of encapsulated bacteria including H. influenzae serotype b (Hib) and Streptococcus pneumoniae (e.g., serotype 3 and 4). Removal of the capsule, as exemplified by Hib RM804, resulted in increased EF-Tu surface density. Finally, anti-NTHi EF-Tu IgG promoted complement-dependent bacterial killing of NTHi and other unencapsulated Gram-negative bacteria as well as opsonophagocytosis of Gram-positive bacteria. In conclusion, our data demonstrate that NTHi EF-Tu is surface-exposed and recognized by antibodies mediating host innate immunity against NTHi in addition to other unencapsulated respiratory tract bacteria.
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