A lipid zipper triggers bacterial invasion

Glycosphingolipids are important structural constituents of cellular membranes. They are involved in the formation of nanodomains (“lipid rafts”), which serve as important signaling platforms. Invasive bacterial pathogens exploit these signaling domains to trigger actin polymerization for the bending of the plasma membrane and the engulfment of the bacterium—a key process in bacterial uptake. However, it is unknown whether glycosphingolipids directly take part in the membrane invagination process. Here, we demonstrate that a “lipid zipper,” which is formed by the interaction between the bacterial surface lectin LecA and its cellular receptor, the glycosphingolipid Gb3, triggers plasma membrane bending during host cell invasion of the bacterium Pseudomonas aeruginosa. In vitro experiments with Gb3-containing giant unilamellar vesicles revealed that LecA/Gb3-mediated lipid zippering was sufficient to achieve complete membrane engulfment of the bacterium. In addition, theoretical modeling elucidated that the adhesion energy of the LecA–Gb3 interaction is adequate to drive the engulfment process. In cellulo experiments demonstrated that inhibition of the LecA/Gb3 lipid zipper by either lecA knockout, Gb3 depletion, or application of soluble sugars that interfere with LecA binding to Gb3 significantly lowered P. aeruginosa uptake by host cells. Of note, membrane engulfment of P. aeruginosa occurred independently of actin polymerization, thus corroborating that lipid zippering alone is sufficient for this crucial first step of bacterial host-cell entry. Our study sheds new light on the impact of glycosphingolipids in the cellular invasion of bacterial pathogens and provides a mechanistic explication of the initial uptake processes.Glycosphingolipids (GSLs) are essential components of biological membranes that have significant impact on the organization and the molecular architecture of the membrane (1, 2). Preferential association of GSLs with cholesterol, various other lipid species, and proteins induces formation of micro- and nanodomains. Such domains, which are also termed “lipid rafts,” are involved in the signal transduction across the membrane (3). Several invasive bacterial pathogens hijack these membrane domains as signaling platforms for actin polymerization, which leads to the engulfment of the bacterium by the host plasma membrane (PM) (4–6). For example, Listeria monocytogenes exploits lipid raft-dependent E-cadherin and HGF-R/Met signaling in the host cell to trigger actin polymerization for bacterial uptake (7). The opportunistic bacterium Pseudomonas aeruginosa can also invade and survive in diverse epithelial and endothelial cells (8–14), which significantly contributes to its pathogenicity (8, 10, 15). Numerous reports suggested various host cell factors, including cell surface receptors and signaling components, including α₅β₁ integrin, cystic fibrosis transmembrane conductance regulator, and Abelson tyrosine-protein kinase 1-dependent pathway (16–18), that are involved in the cellular uptake of P. aeruginosa. Host-cell GSLs have been identified as important molecules contributing to host specificity and adhesion of P. aeruginosa (19, 20). Recent observations suggest that GSLs might be of critical importance for the internalization of P. aeruginosa into nonphagocytic cells (9). The homotetrameric, galactophilic lectin LecA, which is localized to the outer bacterial membrane (21), belongs to the carbohydrate binding proteins expressed by P. aeruginosa that recognize GSLs (22, 23) and represents one of the virulence factors (24). The preferential binding of LecA to the GSL globotriaosylceramide (also known as Gb3/CD77 or Pk histo-blood group antigen) (22, 25) prompted us to investigate the role of LecA–Gb3 interaction in the cellular uptake of P. aeruginosa.In our study, we demonstrated that GSLs have a major impact on the bending of the host plasma membrane and engulfment of the pathogen. Binding of GSL by P. aeruginosa induced negative membrane curvature on synthetic, giant unilamellar vesicles (GUVs), solely driven by LecA–Gb3 interaction. This interaction resulted in a bacterium tightly engulfed by the lipid bilayer of the GUV. Because the glycolipid component plays the major role in this process, we termed this mechanism “lipid zipper” as a novel zipper-type mode of bacterial entry (26). Further experiments revealed that the lipid zipper has a significant impact on P. aeruginosa uptake in several epithelial cell lines. Interestingly, actin polymerization, which was previously identified as a major driving force for membrane deformation and engulfment, was not required for the lipid-triggered process. Moreover, ectopic expression of LecA in Escherichia coli was sufficient to greatly increase its invasion efficiency in Gb3-positive cells.     

The retro-GCN4 leucine zipper sequence forms a stable three-dimensional structure

The question of whether a protein whose natural sequence is inverted adopts a stable fold is still under debate. We have determined the 2. 1-A crystal structure of the retro-GCN4 leucine zipper. In contrast to the two-stranded helical coiled-coil GCN4 leucine zipper, the retro-leucine zipper formed a very stable, parallel four-helix bundle, which now lends itself to further structural and functional studies.     

Homez,a homeobox leucine zipper gene specific to the vertebrate lineage

This work describes a vertebrate homeobox gene, designated Homez (homeodomain leucine zipper-encoding gene), that encodes a protein with an unusual structural organization. There are several regions within Homez, including three atypical homeodomains, two leucine zipper-like motifs, and an acidic domain. The gene is ubiquitously expressed in human and murine tissues, although the expression pattern is more restricted during mouse development. Genomic analysis revealed that human and mouse genes are located at 14q11.2 and 14C, respectively, and are composed of two exons. The zebrafish and pufferfish homologs share high similarity to mammalian sequences, particularly within the homeodomain sequences. Based on homology of homeodomains and on the similarity in overall protein structure, we delineate Homez and members of ZHX family of zinc finger homeodomain factors as a subset within the superfamily of homeobox-containing proteins. The type and composition of homeodomains in the Homez subfamily are vertebrate-specific. Phylogenetic analysis indicates that Homez lineage was separated from related genes >400 million years ago before separation of ray- and lobe-finned fishes. We apply a duplication-degeneration-complementation model to explain how this family of genes has evolved.     

A conserved retina-specific gene encodes a basic motif/leucine zipper domain.

Using subtraction cloning, we have isolated a human cDNA, AS321, which is expressed in retina and retinoblastoma cell lines but not in any other tissue or cell line tested. AS321 mRNA is detected in all cells of neural retina, with a high level of expression in photoreceptors. The polypeptide sequence deduced from the cDNA reveals consensus phosphorylation sites for protein kinase A and proline-directed protein kinase. Its C-terminal region contains a basic motif and a leucine zipper domain similar to the DNA binding proteins of the jun and fos oncoprotein family, and it shows a strong similarity to the product of an avian retroviral oncogene, v-maf. The gene for AS321 is conserved during evolution and is expressed in vertebrate retina. We propose to name the gene NRL (neural retina leucine zipper.     

Use of sternal zipper in open heart surgery]

A sternal zipper was used in 50 patients with an unstable haemodynamic condition after open heart surgery. The patients were 19 women and 31 men (average age 51.6 years, range 7 to 67 years). The indications for surgery were aortocoronary bypass in 25 cases, replacement of the ascending aorta in 7 cases, valve replacement in 16 cases and correction of congenital heart disease in 2 cases. Twenty eight patients required circulatory assistance. The sternal zipper was used for 4 to 72 hours (average 29.5 hours) and mediastinal toilet was performed at least every 24 hours. At each opening of the zipper, 3 bacteriological swabs were taken from 3 different sites in the mediastinum and sent for culture. Global mortality was 36% (N = 18). The cause of death was a low output syndrome in 12 cases, hepatic and renal failure in 2 cases, resistant arrhythmia in 1 case, neurological complication in 1 case and septicaemia in 2 cases. There was one late death 3 months after hospital discharge which was attributed to a cardiac arrhythmia. The sternal zipper would seem to be a valuable option when the operative conditions are difficult, allowing the chest to remain open, so preventing cardiac compression during a critical period.     

Structure-function studies on Escherichia coli MetR protein, a putative prokaryotic leucine zipper protein.

The Escherichia coli metR gene has been sequenced. The sequence predicts a protein of 317 amino acids and a calculated molecular weight of 35,628. This is about 15% larger than the protein from Salmonella typhimurium reported previously [Plamann, L.S. & Stauffer, G.V. (1987) J. Bacteriol. 169, 3932-3937]. The protein is a homodimer and contains a leucine zipper motif characteristic of many eukaryotic DNA-binding proteins. Replacement of two of the leucines in the leucine zipper region of the MetR protein, or substitution of proline for one of the leucines, results in loss of biological activity of the protein. In addition, truncation studies have identified a region on MetR that may be involved in the homocysteine activation of metE expression.     

Structure of homeobox-leucine zipper genes suggests a model for the evolution of gene families.

Homeobox genes are present in both plants and animals. Homeobox-leucine zipper genes, however, have been identified thus far only in the small mustard plant Arabidopsis thaliana. This observation suggests that homeobox-leucine zipper genes evolved after the divergence of plants and animals, perhaps to mediate specific regulatory events. To better understand this gene family, we isolated several sequences containing the homeobox-leucine zipper motif and carried out a comparative analysis of nine homeobox-leucine zipper genes (HAT1, HAT2, HAT3, HAT4, HAT5, HAT7, HAT9, HAT14, and HAT22). Gene structures, sequence comparisons, and chromosomal locations suggest a simple model for the evolution of these genes. The model postulates that a primordial homeobox gene acquired a leucine zipper by exon capture. The nascent homeobox-leucine zipper gene then appears to have undergone a series of gene duplication and chromosomal translocation events, leading to the formation of the HAT gene family. This work has general implications for the evolution of regulatory genes.     

Signal processing by its coil zipper domain activates IKK gamma

NF-kappaB activation occurs upon degradation of its inhibitor I-kappaB and requires prior phosphorylation of the inhibitor by I-kappaB kinase (IKK). Activity of IKK is governed by its noncatalytic subunit IKKgamma. Signaling defects due to missense mutations in IKKgamma have been correlated to its inability to either become ubiquitylated or bind ubiquitin noncovalently. Because the relative contribution of these events to signaling had remained unknown, we have studied mutations in the coil-zipper (CoZi) domain of IKKgamma that either impair signaling or cause constitutive NF-kappaB activity. Certain signaling-deficient alleles neither bound ubiquitin nor were they ubiquitylated by TRAF6. Introducing an activating mutation into those signaling-impaired alleles restored their ubiquitylation and created mutants constitutively activating NF-kappaB without repairing the ubiquitin-binding defect. Constitutive activity therefore arises downstream of ubiquitin binding but upstream of ubiquitylation. Such constitutive activity reveals a signal-processing function for IKKgamma beyond that of a mere ubiquitin-binding adaptor. We propose that this signal processing may involve homophilic CoZi interactions as suggested by the enhanced affinity of CoZi domains from constitutively active IKKgamma.     

Trp zipper folding kinetics by molecular dynamics and temperature-jump spectroscopy

We studied the microsecond folding dynamics of three beta hairpins (Trp zippers 1-3, TZ1-TZ3) by using temperature-jump fluorescence and atomistic molecular dynamics in implicit solvent. In addition, we studied TZ2 by using time-resolved IR spectroscopy. By using distributed computing, we obtained an aggregate simulation time of 22 ms. The simulations included 150, 212, and 48 folding events at room temperature for TZ1, TZ2, and TZ3, respectively. The all-atom optimized potentials for liquid simulations (OPLS(aa)) potential set predicted TZ1 and TZ2 properties well; the estimated folding rates agreed with the experimentally determined folding rates and native conformations were the global potential-energy minimum. The simulations also predicted reasonable unfolding activation enthalpies. This work, directly comparing large simulated folding ensembles with multiple spectroscopic probes, revealed both the surprising predictive ability of current models as well as their shortcomings. Specifically, for TZ1-TZ3, OPLS for united atom models had a nonnative free-energy minimum, and the folding rate for OPLS(aa) TZ3 was sensitive to the initial conformation. Finally, we characterized the transition state; all TZs fold by means of similar, native-like transition-state conformations.     

A Drosophila photoreceptor cell-specific protein, calphotin, binds calcium and contains a leucine zipper.

The calphotin protein, encoded by the calphotin (cap) gene, is expressed in the soma and axons of all Drosophila photoreceptor cells. It is expressed early in photo-receptor cell development, at the time when cell-type decisions are being made. Expression of calphotin is not altered by the glass mutation, which blocks photoreceptor cell development. The calphotin protein binds calcium and contains a long C-terminal leucine zipper. Potential implications of these properties are discussed.     

Development of pilus organelle subassemblies in vitro depends on chaperone uncapping of a beta zipper

The major subassemblies of virulence-associated P pili, the pilus rod (comprised of PapA) and tip fibrillum (comprised of PapE), were reconstituted from purified chaperone-subunit complexes in vitro. Subunits are held in assembly-competent conformations in chaperone-subunit complexes prior to their assembly into mature pili. The PapD chaperone binds, in part, to a conserved motif present at the C terminus of the subunits via a beta zippering interaction. Amino acid residues in this conserved motif were also found to be essential for subunit–subunit interactions necessary for the formation of pili, thus revealing a molecular mechanism whereby the PapD chaperone may prevent premature subunit–subunit interactions in the periplasm. Uncapping of the chaperone-protected C terminus of PapA and PapE was mimicked in vitro by freeze–thaw techniques and resulted in the formation of pilus rods and tip fibrillae, respectively. A mutation in the leading edge of the beta zipper of PapA produces pilus rods with an altered helical symmetry and azimuthal disorder. This change in the number of subunits per turn of the helix most likely reflects involvement of the leading edge of the beta zipper in forming a right-handed helical cylinder. Organelle development is a fundamental process in all living cells, and these studies shed new light on how immunoglobulin-like chaperones govern the formation of virulence-associated organelles in pathogenic bacteria.