scholarly journals Chromosomal Expression of the Haemophilus influenzae Hap Autotransporter Allows Fine-Tuned Regulation of Adhesive Potential via Inhibition of Intermolecular Autoproteolysis

2003 ◽  
Vol 185 (5) ◽  
pp. 1608-1615 ◽  
Author(s):  
Doran L. Fink ◽  
Joseph W. St. Geme
Keyword(s):  
Extracellular Matrix ◽  
Epithelial Cells ◽  
Haemophilus Influenzae ◽  
Critical Role ◽  
Extracellular Matrix Proteins ◽  
Matrix Proteins ◽  
Passenger Domain ◽  
Respiratory Epithelial Cells ◽  
Bacterial Aggregation ◽  
Respiratory Epithelial

ABSTRACT The Haemophilus influenzae Hap autotransporter is a nonpilus adhesin that promotes adherence to respiratory epithelial cells and selected extracellular matrix proteins and facilitates bacterial aggregation and microcolony formation. Hap consists of a 45-kDa outer membrane translocator domain called Hapβ and a 110-kDa extracellular passenger domain called HapS. All adhesive activity resides within HapS, which also contains protease activity and directs its own secretion from the bacterial cell surface via intermolecular autoproteolysis. In the present study, we sought to determine the relationship between the magnitude of Hap expression, the efficiency of Hap autoproteolysis, and the level of Hap-mediated adherence and aggregation. We found that a minimum threshold of Hap precursor was required for autoproteolysis and that this threshold approximated expression of Hap from a chromosomal allele, as occurs in H. influenzae clinical isolates. Chromosomal expression of wild-type Hap was sufficient to promote significant adherence to epithelial cells and extracellular matrix proteins, and adherence was enhanced substantially by inhibition of autoproteolysis. In contrast, chromosomal expression of Hap was sufficient to promote bacterial aggregation only when autoproteolysis was inhibited, indicating that the threshold for Hap-mediated aggregation is above the threshold for autoproteolysis. These results highlight the critical role of autoproteolysis and an intermolecular mechanism of cleavage in controlling the diverse adhesive activities of Hap.

scholarly journals The Haemophilus influenzae Hap Autotransporter Binds to Fibronectin, Laminin, and Collagen IV

2002 ◽  
Vol 70 (9) ◽  
pp. 4902-4907 ◽  
Author(s):  
Doran L. Fink ◽  
Bruce A. Green ◽  
Joseph W. St. Geme
Keyword(s):  
Extracellular Matrix ◽  
Respiratory Tract ◽  
Haemophilus Influenzae ◽  
Extracellular Matrix Proteins ◽  
Collagen Iv ◽  
Binding Domain ◽  
Matrix Proteins ◽  
Heparin Binding ◽  
Upper Respiratory Tract ◽  
Passenger Domain

ABSTRACT Nontypeable Haemophilus influenzae (NTHI) initiates infection by colonizing the upper respiratory tract mucosa. NTHI disease frequently occurs in the context of respiratory tract inflammation, where organisms encounter damaged epithelium and exposed basement membrane. In this study, we examined interactions between the H. influenzae Hap adhesin and selected extracellular matrix proteins. Hap is an autotransporter protein that undergoes autoproteolytic cleavage, with release of the adhesive passenger domain, Haps, from the bacterial cell surface. We found that Hap promotes bacterial adherence to purified fibronectin, laminin, and collagen IV and that Hap-mediated adherence is enhanced by inhibition of autoproteolysis. Adherence is inhibited by pretreatment of bacteria with a polyclonal antiserum recognizing Haps. Purified Haps binds with high affinity to fibronectin, laminin, and collagen IV but not to collagen II. Binding of Haps to fibronectin involves interaction with the 45-kDa gelatin-binding domain but not the 30-kDa heparin-binding domain of fibronectin. Taken together, these observations suggest that interactions between Hap and extracellular matrix proteins may play an important role in NTHI colonization of the respiratory tract.

STAT-3 regulates surfactant phospholipid homeostasis in normal lung and during endotoxin-mediated lung injury

2008 ◽  
Vol 104 (6) ◽  
pp. 1753-1760 ◽  
Author(s):  
Machiko Ikegami ◽  
Angelica Falcone ◽  
Jeffrey A. Whitsett
Keyword(s):  
Epithelial Cells ◽  
Lung Injury ◽  
Critical Role ◽  
Lung Mechanics ◽  
Normal Lung ◽  
Type Ii Cells ◽  
Type Ii ◽  
Phospholipid Synthesis ◽  
Respiratory Epithelial Cells ◽  
Respiratory Epithelial

Acute lung injury associated with surfactant deficiency remains a major cause of pulmonary morbidity and mortality. Since signal transducer and activator of transcription-3 (STAT-3) plays an important role in protecting respiratory epithelial cells during injury, we hypothesized that STAT-3 may regulate gene expression in type II cells that mediate surfactant phospholipid synthesis. Conditional deletion of Stat-3 in respiratory epithelial cells in the lung of transgenic mice ( Stat-3Δ/Δ mice) decreased surfactant phospholipid synthesis and secretion. Deletion of Stat-3 was associated with decreased expression of Akt2, Srebf-1, and other genes expressed in type II cells that may influence surfactant phospholipid synthesis ( Glut-1, Slc34a2, Gpam, Acox2, and Cds2). Stat-3Δ/Δ mice were more susceptible to intratracheal lipopolysaccharide (LPS). Saturated phosphatidylcholine and surfactant protein B levels were significantly decreased in bronchoalveolar lavage fluid from LPS-treated Stat-3Δ/Δ mice. Alveolar capillary leak, proinflammatory cytokine expression, and perturbations of lung mechanics caused by LPS were exacerbated after deletion of STAT-3. STAT-3 plays a critical role in the regulation of surfactant lipid synthesis in the normal lung and during lung injury caused by LPS.

Laminin α-chain expression and basement membrane formation by MLE-15 respiratory epithelial cells

2002 ◽  
Vol 282 (5) ◽  
pp. L1004-L1011 ◽  
Author(s):  
Nguyet M. Nguyen ◽  
Yushi Bai ◽  
Katsumi Mochitate ◽  
Robert M. Senior
Keyword(s):  
Epithelial Cells ◽  
Basement Membrane ◽  
Critical Role ◽  
Basement Membranes ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Type Ii ◽  
Respiratory Epithelial Cells ◽  
Alveolar Type Ii Cells ◽  
Respiratory Epithelial

Basement membranes have a critical role in alveolar structure and function. Alveolar type II cells make basement membrane constituents, including laminin, but relatively little is known about the production of basement membrane proteins by murine alveolar type II cells and a convenient system is not available to study basement membrane production by murine alveolar type II cells. To facilitate study of basement membrane production, with particular focus on laminin chains, we examined transformed murine distal respiratory epithelial cells (MLE-15), which have many structural and biochemical features of alveolar type II cells. We found that MLE-15 cells produce laminin-α5, a trace amount of laminin-α3, laminins-β1 and -γ1, type IV collagen, and perlecan. Transforming growth factor-β1 significantly induces expression of laminin-α1. When grown on a fibroblast-embedded collagen gel, MLE-15 cells assemble a basement membrane-like layer containing laminin-α5. These findings indicate that MLE-15 cells will be useful in modeling basement membrane production and assembly by alveolar type II cells.

scholarly journals Interaction of Fimbriae of Haemophilus influenzae Type B with Heparin-Binding Extracellular Matrix Proteins

2000 ◽  
Vol 68 (10) ◽  
pp. 5696-5701 ◽  
Author(s):  
Ritva Virkola ◽  
Mirko Brummer ◽  
Heikki Rauvala ◽  
Loek van Alphen ◽  
Timo K. Korhonen
Keyword(s):  
Extracellular Matrix ◽  
Haemophilus Influenzae ◽  
Extracellular Matrix Proteins ◽  
Matrix Proteins ◽  
Haemophilus Influenzae Type ◽  
Heparin Binding ◽  
Type B ◽  
Binding Domains ◽  
Human Fibronectin ◽  
High Concentrations

ABSTRACT The interaction of the fimbriae of Haemophilus influenzae type b (Hib) with two heparin-binding extracellular matrix proteins, human fibronectin (Fn) and heparin-binding growth-associated molecule (HB-GAM) from mouse, were studied. The fimbriated Hib strain 770235 fim+, as well as the recombinant strainE. coli HB101(pMH140), which expressed Hib fimbriae, adhered strongly to Fn and HB-GAM immobilized on glass. Purified Hib fimbriae bound to Fn and HB-GAM, and within the Fn molecule, the binding was localized to the N-terminal 30,000-molecular-weight (30K) and 40K fragments, which contain heparin-binding domains I and II, respectively. Fimbrial binding to Fn, HB-GAM, and the 30K and the 40K fragments was inhibited by high concentrations of heparin. The results show that fimbriae of Hib interact with heparin-binding extracellular matrix proteins. The nonfimbriated Hib strain 770235 fim− exhibited a low level of adherence to Fn but did not react with HB-GAM, indicating that Hib strains also possess a fimbria-independent mechanism to interact with Fn.

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