Browsing by Subject "bacterial pathogenesis"
Results Per Page
Sort Options
Item Open Access Cha-Cha-Cha: Variable Adhesive Activity of the Haemophilus Cryptic Genospecies Trimeric Autotransporter Cha(2009) Sheets, Amanda JoanDisease caused by the Gram-negative Haemophilus cryptic genospecies begins with colonization of the maternal genital or neonatal respiratory tract. The primary goal of this work was to identify and characterize the molecular determinant(s) of Haemophilus cryptic genospecies adherence as a means to better understand the specific adaptation of this species to the urogenital tract and neonatal respiratory tract. Using transposon mutagenesis of prototype strain 1595, we identified a locus that is essential for Haemophilus cryptic genospecies adherence to a variety of epithelial cell lines of both genital and respiratory origin. This locus encodes a protein called Cha that shares homology with trimeric autotransporters. Trimeric autotransporters are composed of an N-terminal signal peptide, an internal passenger domain that harbors adhesive activity, and a short C-terminal membrane anchor domain and are classically characterized by head-stalk-anchor domain architecture. By generating chimeric proteins, we demonstrated that the C-terminus of Cha trimerizes in the bacterial outer membrane and is capable presenting a heterologous passenger domain (Hia) in a functional form, thus confirming that Cha is a trimeric autotransporter. Southern analysis revealed that cha is unique to the Haemophilus cryptic genospecies and is ubiquitous among these strains.
Similar to a number of trimeric autotransporters, the passenger domain of Cha contains scattered clusters of YadA-like head domains associated with head-to-stalk neck adaptor motifs, predicted coiled-coil stalks and a series of identical tandem coding repeats which are not required for adherence. By evaluating the adherence capacity of H. influenzae expressing Cha deletion derivatives, we established that the N-terminal 473 residues of Cha harbor the binding domains responsible for Cha-mediated adherence to epithelial cells. In additional studies, we demonstrated that this same N-terminal region mediates bacterial aggregation through inter-bacterial Cha-Cha binding.
Further analysis revealed that variable Cha-mediated adherence is linked to spontaneous changes in the number of identical tandem repeats predicted to comprise a coiled-coil stalk domain. Variation in repeat copy number has a direct effect on Cha adhesive and aggregative activity, independent of an impact on transcription of the cha locus or surface localization of Cha protein. Moreover, length of Cha surface fibers correlates with repeat copy number expansion. We propose two hypotheses to explain how repeat expansion inhibits bacterial aggregation and host cell binding: 1) Variation in the number of 28-amino acid repeats may influence the conformation of Cha, thus changing the surface accessibility of the Cha binding pocket. 2) Repeat expansion results in the formation of long, flexible Cha fibers on the bacterial cell surface that may have a greater propensity to interact with neighboring Cha trimers at the N-terminus, thereby precluding adherence to other bacteria or host epithelial cells.
In additional studies screening adherent cryptic genospecies isolates for expression of Cha protein, we identified an additional, antigenically-divergent Cha variant that we refer to as Cha2. Amino acid sequence and domain comparison of Cha2 with Cha (now Cha1) revealed that the structurally undefined N-terminal sequences (encompassing the Cha1 adhesive and aggregative domain) are strikingly divergent. Inspite of this, Cha2 mediates efficient adherence to human epithelial cells, similar to Cha1.
Identification of Cha offers insight into the apparent tissue tropism associated with the Haemophilus cryptic genospecies. We speculate that the unique regulation of Cha adhesive activity enhances the adaptive capability of this pathogenic organism in the human host.
Item Open Access Characterization of Peripheral-Membrane Enzymes Required for Lipid A Biosynthesis in Gram-Negative Bacteria(2010) Metzger, Louis EugeneGram-negative bacteria possess an asymmetric outer membrane in which the inner leaflet is composed primarily of phospholipids while the outer leaflet contains both phospholipids and lipopolysaccharide (LPS). LPS forms a structural barrier that protects Gram-negative bacteria from antibiotics and other environmental stressors. The lipid A anchor of LPS is a glucosamine-based saccharolipid that is further modified with core and O-antigen sugars. In addition to serving a structural role as the hydrophobic anchor of LPS, lipid A is recognized by the innate immune system in animal cells and macrophages. The enzymes of Lipid A biosynthesis are conserved in Gram-negative bacteria; in most species, a single copy of each bio-synthetic gene is present. The exception is lpxH, which is an essential gene encoding a membrane-associated UDP-2,3-diacylglucosamine hydrolase, which catalyzed the attack of water upon the alpha-phosphate of its substrate and the leaving of UMP, resulting in the formation of lipid X. Many Gram-negatives lack an lpxH orthologue, yet these species must possess an activity analogous to that of LpxH. We used bioinformatics approaches to identify a candidate gene, designated lpxI, encoding this activity in the model organism Caulobacter crescentus. We then demonstrated that lpxI can rescue Escherichia coli deficient in lpxH. Moreover, we have shown that LpxI possesses robust and specific UDP-2,3-diacylglucosamine hydrolase activity in vitro. We have developed high-yield purification schema for Caulobacter crescentus LpxI (CcLpxI) heterologously expressed in E. coli. We crystallized CcLpxI and determined its 2.6 Å x-ray crystal structure in complex with lipid X. CcLpxI, which has no known homologues, consists of two novel domains connected by a linker. Moreover, we have identified a point mutant of CcLpxI which co-purifies with its substrate in a 0.85:1 molar ratio. We have solved the x-ray crystal structure of this mutant to 3.0 Å; preliminary comparison with the product-complexed model reveals striking differences. The findings described herein set the stage for further mechanistic and structural characterization of this novel enzyme.
In this work, we also isolate and characterize LpxB, an essential lipid A biosynthetic gene which is conserved among all Gram-negative bacteria. We purify E. coli and Hemophilus influeznea LpxB to near-homogeneity on a 10 mg scale, and we determine that E. coli LpxB activity is dependent upon the bulk surface concentration of its substrates in a mixed micellar assay system, suggesting that catalysis occurs at the lipid interface. E. coli LpxB partitions with membranes, but this interaction is partially abolished in high-salt conditions, suggesting that a significant component of LpxB's membrane association is ionic in nature. E. coli LpxB (Mr ~ 43 kDa) is a peripheral membrane protein, and we demonstrate that it co-purifies with phospholipids. We estimate, by autoradiography and mass-spectrometry, molar ratios of phospholipids to purified enzyme of 1.6-3.5:1. Transmission electron microscopy reveals the accumulation of intra-cellular membranes when LpxB is massively over-expressed. Alanine-scanning mutagenesis of selected conserved LpxB residues identified two, D89A and R201A, for which no residual catalytic activity is detected. Our data support the hypothesis that LpxB performs catalysis at the cytoplasmic surface of the inner membrane, and provide a rational starting-point for structural studies. This work contributes to knowledge of the small but growing set of structurally and mechanistically characterized enzymes which perform chemistry upon lipids.