| 1. |
- Ünal, Can, et al.
(författare)
-
QseC controls biofilm formation of non typeable Haemophilus influenzae in addition to an A1-2 dependent mechanism
- 2012
-
Ingår i: International Journal of Medical Microbiology. - : Elsevier. - 1438-4221 .- 1618-0607. ; 302:6, s. 261-269
-
Tidskriftsartikel (refereegranskat)abstract
- Non-typeable Haemophilus influenzae (NTHi) is a common pathogen associated with diseases such as acute otitis media or exacerbations in patients with chronic obstructive pulmonary disease. The biofilm-forming capability substantially contributes to the persistence of NTHi. However, the regulation of biofilm formation is not completely understood. Quorum sensing regulated by autoinducer-2 produced by luxS is until now the only described regulatory mechanism. In this study, we show that the two-component signalling system QseB/C is involved in the biofilm formation of NTHi in vitro. An isogenic NTHi mutant of qseC (Hi3655KR2) showed a significant decrease in biofilm formation under static and semi-static conditions as assessed by crystal violet staining. In addition, under constant flow conditions, Hi3655KR2 formed less biofilm after 48 h. The biofilm defects were irrespective of autoinducer-2 levels. Hence, here we suggest for the first time a regulatory circuit in NTHi, which controls biofilm formation by mechanisms other than or in addition to luxS-dependent factors.
|
|
| 2. |
- Barfod, Anders, et al.
(författare)
-
In Vitro Selection of RNA Aptamers Directed Against Protein E: A Haemophilus influenzae Adhesin. : a Haemophilus influenzae adhesin
- 2014
-
Ingår i: Molecular Biotechnology. - : Springer Science and Business Media LLC. - 1559-0305 .- 1073-6085. ; 56:8, s. 714-725
-
Tidskriftsartikel (refereegranskat)abstract
- Protein E (PE) of Haemophilus influenzae is a highly conserved ubiquitous surface protein involved in adhesion to and activation of epithelial cells. The host proteins-vitronectin, laminin, and plasminogen are major targets for PE-dependent interactions with the host. To identify novel inhibitory molecules of PE, we used an in vitro selection method based on systematic evolution of ligands by exponential enrichment known as SELEX in order to select 2'F-modified RNA aptamers that specifically bind to PE. Fourteen selection cycles were performed with decreasing concentrations of PE. Sequencing of clones from the 14th selection round revealed the presence of semiconserved sequence motifs in loop regions of the RNA aptamers. Among these, three aptamers showed the highest affinity to PE in electrophoretic mobility shift assays and in dot blots. These three aptamers also inhibited the interaction of PE with vitronectin as revealed by ELISA. Moreover, pre-treatment of H. influenzae with the aptamers significantly inhibited binding of vitronectin to the bacterial surface. Biacore experiments indicated that one of the aptamers had a higher binding affinity for PE as compared to the other aptamers. Our results show that it is possible to select RNA inhibitors against bacterial adhesins using SELEX in order to inhibit interactions with target proteins.
|
|
| 3. |
- Barthel, Diana, et al.
(författare)
-
Haemophilus influenzae Uses the Surface Protein E To Acquire Human Plasminogen and To Evade Innate Immunity
- 2012
-
Ingår i: Journal of Immunology. - : The American Association of Immunologists. - 1550-6606 .- 0022-1767. ; 188:1, s. 379-385
-
Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Pathogenic microbes acquire the human plasma protein plasminogen to their surface. In this article, we characterize binding of this important coagulation regulator to the respiratory pathogen nontypeable Haemophilus influenzae and identify the Haemophilus surface protein E (PE) as a new plasminogen-binding protein. Plasminogen binds dose dependently to intact bacteria and to purified PE. The plasminogen-PE interaction is mediated by lysine residues and is also affected by ionic strength. The H. influenzae PE knockout strain (nontypeable H. influenzae 3655 Delta pe) bound plasminogen with similar to 65% lower intensity as compared with the wild-type, PE-expressing strain. In addition, PE expressed ectopically on the surface of Escherichia coli also bound plasminogen. Plasminogen, either attached to intact H. influenzae or bound to PE, was accessible for urokinase plasminogen activator. The converted active plasmin cleaved the synthetic substrate S-2251, and the natural substrates fibrinogen and C3b. Using synthetic peptides that cover the complete sequence of the PE protein, the major plasminogen-binding region was localized to a linear 28-aa-long N-terminal peptide, which represents aa 41-68. PE binds plasminogen and also vitronectin, and the two human plasma proteins compete for PE binding. Thus, PE is a major plasminogen-binding protein of the Gram-negative bacterium H. influenzae, and when converted to plasmin, PE-bound plasmin aids in immune evasion and contributes to bacterial virulence. The Journal of Immunology, 2012, 188: 379-385.
|
|
| 4. |
- Hallstrom, Teresia, et al.
(författare)
-
Haemophilus influenzae Protein E Binds to the Extracellular Matrix by Concurrently Interacting With Laminin and Vitronectin
- 2011
-
Ingår i: Journal of Infectious Diseases. - : Oxford University Press (OUP). - 1537-6613 .- 0022-1899. ; 204:7, s. 1065-1074
-
Tidskriftsartikel (refereegranskat)abstract
- Nontypeable Haemophilus influenzae (NTHi) causes otitis media and is commonly found in patients with chronic obstructive pulmonary disease (COPD). Adhesins are important for bacterial attachment and colonization. Protein E (PE) is a recently characterized ubiquitous 16 kDa adhesin with vitronectin-binding capacity that results in increased survival in serum. In addition to PE, NTHi utilizes Haemophilus adhesion protein (Hap) that binds to the basement-membrane glycoprotein laminin. We show that most clinical isolates bind laminin and that both Hap and PE are crucial for the NTHi-dependent interaction with laminin as revealed with different mutants. The laminin-binding region is located at the N-terminus of PE, and PE binds to the heparin-binding C-terminal globular domain of laminin. PE simultaneously attracts vitronectin and laminin at separate binding sites, proving the multifunctional nature of the adhesin. This previously unknown PE-dependent interaction with laminin may contribute to NTHi colonization, particularly in smokers with COPD.
|
|
| 5. |
- Jalalvand, Farshid, et al.
(författare)
-
Haemophilus influenzae Protein F Mediates Binding to Laminin and Human Pulmonary Epithelial Cells.
- 2012
-
Ingår i: Journal of Infectious Diseases. - : Oxford University Press (OUP). - 1537-6613 .- 0022-1899. ; 207:5, s. 803-813
-
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.
|
|
| 6. |
- Singh, Birendra, et al.
(författare)
-
A fine-tuned interaction between the trimeric autotransporter Haemophilus surface fibrils and vitronectin leads to serum resistance and adherence to respiratory epithelial cells.
- 2014
-
Ingår i: Infection and Immunity. - 1098-5522. ; 82:6, s. 2378-2389
-
Tidskriftsartikel (refereegranskat)abstract
- Haemophilus influenzae type b (Hib) escapes the host immune system by recruitment of the complement regulator vitronectin that inhibits the formation of the membrane attack complex (MAC) by inhibiting C5b-C7 complex formation and C9 polymerization. We previously reported that Hib acquires vitronectin at the surface by using Haemophilus surface fibrils (Hsf). Here we studied in detail the interaction between Hsf and vitronectin and its role in inhibition of MAC formation and invasion of lung epithelial cells. The vitronectin-binding region of Hsf was defined at the N-terminal comprising amino acids Hsf 429-652. Moreover, the Hsf recognition site on vitronectin consisted of the C-terminal amino acids 352-374. H. influenzae was killed more rapidly in vitronectin-depleted serum when compared to normal human serum (NHS), and an increased MAC deposition was observed at the surface of an Hsf-deficient H. influenzae mutant. In parallel, Hsf-expressing E. coli selectively acquired vitronectin from serum that resulted in significant inhibition of the MAC. Moreover, when vitronectin was bound to Hsf an increased bacterial adherence and internalization of epithelial cells was observed. Taken together, we have defined a fine-tuned protein-protein interaction between Hsf and vitronectin that may contribute to an increased virulence of Hib.
|
|
| 7. |
- Singh, Birendra, et al.
(författare)
-
Crystallization and X-ray diffraction analysis of a novel surface-adhesin protein: protein E from Haemophilus influenzae.
- 2012
-
Ingår i: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications. - 2053-230X. ; 68:Pt 2, s. 222-226
-
Tidskriftsartikel (refereegranskat)abstract
- Protein E (PE) is a ubiquitous multifunctional surface protein of Haemophilus spp. and other bacterial pathogens of the Pasteurellaceae family. H. influenzae utilizes PE for attachment to respiratory epithelial cells. In addition, PE interacts directly with plasminogen and the extracellular matrix (ECM) components vitronectin and laminin. Vitronectin is a complement regulator that inhibits the formation of the membrane-attack complex (MAC). PE-mediated vitronectin recruitment at the H. influenzae surface thus inhibits MAC and protects against serum bactericidal activity. Laminin is an abundant ECM protein and is present in the basement membrane that helps in adherence of H. influenzae during colonization. Here, the expression, purification and crystallization of and the collection of high-resolution data for this important H. influenzae adhesin are reported. To solve the phase problem for PE, Met residues were introduced and an SeMet variant was expressed and crystallized. Both native and SeMet-containing PE gave plate-like crystals in space group P2(1), with unit-cell parameters a = 44, b = 57, c = 61 Å, β = 96°. Diffraction data collected from native and SeMet-derivative crystals extended to resolutions of 1.8 and 2.6 Å, respectively.
|
|
| 8. |
- Singh, Birendra, et al.
(författare)
-
Haemophilus influenzae protein E recognizes the C-terminal domain of vitronectin and modulates the membrane attack complex.
- 2011
-
Ingår i: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 81, s. 80-98
-
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.
|
|
| 9. |
- Singh, Birendra, et al.
(författare)
-
Human pathogens utilize host extracellular matrix proteins laminin and collagen for adhesion and invasion of the host.
- 2012
-
Ingår i: FEMS Microbiology Reviews. - : Oxford University Press (OUP). - 1574-6976. ; 36:6, s. 1122-1180
-
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.
|
|
| 10. |
- Singh, Birendra, et al.
(författare)
-
Protein E of Haemophilus influenzae Is a Ubiquitous Highly Conserved Adhesin.
- 2010
-
Ingår i: Journal of Infectious Diseases. - : Oxford University Press (OUP). - 1537-6613 .- 0022-1899. ; 201, s. 414-419
-
Tidskriftsartikel (refereegranskat)abstract
- Protein E (PE) of nontypeable Haemophilus influenzae (NTHi) is involved in adhesion and activation of epithelial cells. A total of 186 clinical NTHi isolates, encapsulated H. influenzae, and culture collection strains were analyzed. PE was highly conserved in both NTHi and encapsulated H. influenzae (96.9%-100% identity without the signal peptide). PE also existed in other members of the genus Pasteurellaceae. The epithelial cell binding region (amino acids 84-108) was completely conserved. Phylogenetic analysis of the pe sequence separated Haemophilus species into 2 separate clusters. Importantly, PE was expressed in 98.4% of all NTHi (126 isolates) independently of the growth phase.
|
|
