Browsing by Subject "autotransporter"
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Item Open Access A Tale of Two Proteins: Insights into the Haemophilus influenzae Hap and Hia Autotransporters(2011) Spahich, Nicole AnnNontypeable Haemophilus influenzae (NTHi) is a common commensal in the human nasopharynx that can cause localized respiratory tract diseases such as otitis media, bronchitis, and pneumonia. NTHi adheres to respiratory epithelial cells, a critical step in the process of colonization enabled by bacterial surface adhesive structures called adhesins. One group of NTHi adhesins are autotransporters, proteins that have an N-terminal signal sequence, a C-terminal β-barrel domain, and an internal passenger domain with effector function. The goal of this work was to increase our understanding of two NTHi autotransporters, Hap and Hia.
Hap is a monomeric autotransporter that mediates adherence to epithelial cells and extracellular matrix (ECM) proteins. Hap also self-associates with protein on neighboring bacteria, resulting in bacterial aggregation and microcolony formation. The Hap passenger domain contains the regions responsible for adhesive activity. To define the molecular mechanism of Hap adhesive activity, we crystallized the Hap passenger domain. Characterization of the crystal structure revealed an N-terminal globular domain and a more ordered, prism-like C-terminal domain. Interestingly, Hap crystallized as a multimer, suggesting that Hap-Hap interactions occurred in the passenger domain. Progressive deletions of the β-loops that comprise the C-terminal region disrupted Hap-Hap interactions and led to a defect in bacterial settling. To further support that the C-terminal domain was responsible for Hap-Hap interactions,
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we purified the wild type and truncated passenger domains and conjugated the proteins to latex beads. By light microscopy we visualized bead aggregation when the wild type passenger domain was conjugated to the beads, but not when the truncated passenger domain was conjugated. These results show that the C-terminal portion of the Hap passenger domain is responsible for Hap-Hap interactions leading to multimerization. Hap multimerization could be important in microcolony formation that leads to biofilm formation in vivo.
The ECM binding domain in located in the final 511 amino acids of the Hap passenger domain. To pin-point the region of the ECM protein fibronectin that is recognized by Hap, we spotted small fragments of fibronectin onto nitrocellulose membranes and incubated the membrane with purified Hap passenger domain. Far Western analysis using Hap antibody revealed that the smallest fibronectin region necessary for binding was comprised of the first two type III repeats, FNIII(1-2). To define the regions of Hap responsible for interaction with fibronectin, we mutated motifs in the Hap passenger domain that are important for fibronectin binding in other bacterial proteins. Based on assessment by ELISA, many of the mutations located between amino acids 525-725 caused reduced bacterial binding to fibronectin. However, no mutation totally ablated binding, suggesting that a larger Hap region is involved in fibronectin binding.
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In an additional study, we identified a relationship between Hap levels in the outer membrane and the expression of lipopolysaccharide (LPS) biosynthesis enzymes. Through Western and qPCR analysis, we found that mutation of the rfaF, pgmB, lgtC, kfiC, orfE, rfbP, lsgB and lsgD genes involved in the synthesis of LPS oligosaccharide core in H. influenzae strain Rd/HapS243A resulted in loss of Hap in the bacterial outer membrane and a decrease in hap transcript. In contrast, the same mutations had no effect on outer membrane localization of H. influenzae P5 and IgA1 protease or levels of the p5 or iga1 transcripts, suggesting a Hap-specific effect. Elimination of the HtrA periplasmic protease resulted in a return of Hap to the outer membrane and restoration of wild type levels of hap transcript. We speculate that the lack of certain LPS biosynthesis enzymes causes Hap to mislocalize and accumulate in the periplasm, where it is degraded by HtrA. This degradation then leads to a decrease in hap transcript. lgtC is one of several phase variable LPS biosynthesis genes. Using an antibody against the epitope formed in part by the lgtC gene product, we identified lgtC phase-off bacteria by Western analysis of colony blots. Consistent with our previous observations, in lgtC phase off bacteria Hap was absent from the outer membrane and hap transcript was reduced. By analyzing a lgtC/lic2A double mutant, we found that Hap localization in the outer membrane and hap transcript levels were not related to LPS size but instead to the functions of the LPS synthesis enzymes themselves. This relationship could be beneficial to bacteria in vivo as a way to regulate Hap expression.
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Early models suggested that autotransporters do not require accessory factors for folding and OM insertion. However, mounting recent evidence has suggested that the Bam complex is required for OM localization of most β-barrel proteins, including autotransporters. We studied the role of the Bam complex in OM localization of the trimeric autotransporter Hia. We expressed Hia in E. coli strains with mutations in the Bam complex and found that BamA and BamD were needed for Hia localization, while BamB, BamC, and BamE were not necessary. In further studies, we mutated the C-terminus of Hia and found that the final and third-to-last amino acids were the most important for outer membrane localization.
In summary, this work provides insights into the regulation and adhesive activity of Hap and the outer membrane localization of Hia. We have learned important details about these factors that shed light on aspects of H. influenzae disease and could lead to new antimicrobial therapies.
Item Open Access Insights Into the Virulence Determinants of the Emerging Pathogen Kingella kingae(2012) Porsch, Eric AllenKingella kingae is an emerging bacterial pathogen that is being recognized increasingly as an important etiology of septic arthritis, osteomyelitis, and bacteremia, especially in young children. The pathogenesis of K. kingae disease begins with bacterial adherence to respiratory epithelium in the posterior pharynx. Previous work identified type IV pili as a critical factor for adherence to human epithelial cells. However, the finding that a significant percentage of pharyngeal isolates are non-piliated suggests that K. kingae expresses additional surface factors that modulate interactions with host cells and likely play key roles in the pathogenesis of K. kingae disease. The purpose of this work was to increase our understanding of K. kingae virulence determinants, specifically focused on defining the surface factors and the mechanism involved in K. kingae adhesive interactions with epithelial cells. Additionally, this work aimed to further characterize components of the K. kingae type IV pilus system, namely the PilC proteins and PilA2.
We first set out to identify non-pilus factors that influence K. kingae interactions with human epithelial cells. Using targeted genetic approaches, we found that insertional inactivation of the gene encoding a predicted trimeric autotransporter protein called Knh (Kingella NhhA homolog) resulted in reduced adherence to human epithelial cells. In addition, using a variety of techniques, including morphological analysis, cationic ferritin staining, and thin section transmission electron microscopy, we established that K. kingae elaborates a surface-associated polysaccharide capsule that requires a predicted ABC-type transporter export operon called ctrABCD for surface presentation. Furthermore, using quantitative human epithelial cell adherence assays, we discovered that the presence of surface capsule interferes with Knh-mediated adherence by non-piliated organisms and that maximal adherence in the presence of capsule requires the predicted type IV pilus retraction machinery, PilT/PilU. Based on the data presented here, we propose a novel adherence mechanism that allows K. kingae to adhere efficiently to human epithelial cells while remaining encapsulated and more resistant to immune clearance.
Having established that K. kingae produces a capsule, a large-scale polysaccharide purification technique was developed for capsule analysis of strain 269-492. Biochemical assays determined that the purified material contained thiobarbituric and phenol-sulfuric acid reactive glycosyl residues. In collaboration with the University of Georgia Complex Carbohydrate Research Center (CCRC), mass spectrometry identified galactose, N-acetyl-galactosamine, and Kdo as the major glycosyl components of the polysaccharide preparation. NMR spectroscopy revealed that the purified material contained two distinct polysaccharides with the structures of →5)–β–Galf–(1→ and →3)–β–GalNAcp–(1→5)–β–Kdop–(2→. Further characterization of the polysaccharides expressed by K. kingae may have implications for disease prevention strategies.
Previous work in our lab found that two PilC-like proteins called PilC1 and PilC2 influence type IV pili expression and pilus-mediated adherence. Production of either PilC1 or PilC2 is necessary for K. kingae piliation and bacterial adherence. We set out to further investigate the role of PilC1 and PilC2 in type IV pilus-associated phenotypes. We found that PilC1 contains a functional nine amino acid calcium-binding (Ca-binding) site with homology to the Pseudomonas aeruginosa PilY1 Ca-binding site and that PilC2 contains a functional 12 amino acid Ca-binding site with homology to the human calmodulin Ca-binding site. Using targeted mutagenesis to disrupt the Ca-binding sites, we demonstrated that the PilC1 and PilC2 Ca-binding sites are dispensable for piliation. Interestingly, we show that the PilC1 site is necessary for twitching motility and adherence to Chang epithelial cells, while the PilC2 site has only a minor influence on twitching motility and no influence on adherence. These findings establish key differences in PilC1 and PilC2 function in K. kingae and provide insights into the biology of the PilC-like family of proteins.
Lastly, we set out to define the role of the PilA2 minor pilin in K. kingae strain 269-492. While previous studies indicated that PilA2 is not essential for pilus expression or adherence to epithelial cells, analysis of the pilin locus in a diverse set of clinical isolates revealed that the pilA2 gene sequence is highly conserved, suggesting it serves an important function. Using targeted mutagenesis we showed that PilA2 is not essential for twitching motility and may or may not be involved in natural competence. Western blot analysis was unable to detect PilA2 in wild type pilus preparations, indicating that it is expressed at a level beneath the assay detection limit or does not localize to the pilus. Additionally, site-directed mutagenesis was used to place pilA2 under control of the highly active pilA1 promoter and showed that PilA2 is able to be assembled into fibers that mediate intermediate adherence to epithelial cells.
Taken together, this work expands our knowledge of the K. kingae surface factor repertoire and provides insights into the roles of type IV pilus components. The mechanism of K. kingae adherence to epithelial cells is beginning to emerge. These contributions may lead to novel strategies for the prevention of invasive K. kingae disease in young children.