基于蛋白质跨外膜自转运系统的细菌细胞表面蛋白展示技术研究进展

革兰氏阴性菌Ⅴ型分泌系统是细菌病原蛋白分泌的主要途径之一,可分为Ⅴa-Ⅴe5个亚型,其中Ⅴa型(即经典的单体自转运蛋白)是细菌毒力和黏附因子向细胞外分泌的重要工具,其在内膜Sec易位子和外膜BAM蛋白复合体的协助下,通过2个连续的跨膜步骤介导蛋白质穿过阴性菌的内外膜.据信Va型是目前已知蛋白质跨膜转运时最简单的分泌途径...     

The Escherichia coli biofilm-promoting protein Antigen 43 does not contribute to intestinal colonization

Abstract Escherichia coli is a versatile organism capable of causing a variety of intestinal and extraintestinal diseases, as well as existing as part of the commensal flora. A variety of factors permit specific attachment to host receptors including fimbrial adhesins and outer membrane proteins such as autotransporters. One of the better characterized autotransporters is Antigen 43 (Ag43), the major phase-variable surface protein of E. coli. Ag43 is associated with bacterial cell-cell aggregation and biofilm formation. Nevertheless, the precise biological significance and contribution to intestinal colonization remain to be elucidated. Here we investigated the contribution of Ag43 to E. coli adherence to intestinal epithelial cells and colonization of the mouse intestine. These investigations revealed that Ag43 increased in vitro adherence of E. coli to epithelial cells by promoting bacterial cell-cell aggregation but that Ag43 did not promote specific interactions with the mammalian cells. Furthermore, Ag43 did not contribute significantly to colonization of the mouse intestine and expression of Ag43 was lost a few days after colonization of the mouse was established. Unexpectedly, considering its similarity to other adhesins, our findings suggest that Ag43 does not act as a direct colonization factor by binding to mammalian cells.     

Molecular evolution of rickettsia surface antigens: evidence of positive selection

The Rickettsia genus is a group of obligate intracellular parasitic alpha-proteobacteria that includes human pathogens responsible for the typhus disease and various types of spotted fevers. rOmpA and rOmpB are two members of the "surface cell antigen" (Sca) autotransporter (AT) protein family that may play key roles in the adhesion of the Rickettsia cells to the host tissue. These molecules are likely determinants for the pathogenicity of the Rickettsia and represent good candidates for vaccine development. We identified the 17 members of this family of outer-membrane proteins in nine fully sequenced Rickettsia genomes. The typical architecture of the Sca proteins is composed of an N-terminal signal peptide and a C-terminal AT domain that promote the export of the central passenger domain to the outside of the bacteria. A characteristic of this family is the frequent degradation of the genes, which results in different subsets of the sca genes being expressed among Rickettsia species. Here, we present a detailed analysis of their phylogenetic relationships and evolution. We provide strong evidence that rOmpA and rOmpB as well as three other members of the Sca protein family--Sca1, Sca2, and Sca4--have evolved under positive selection. The exclusive distribution of the predicted positively selected sites within the passenger domains of these proteins argues that these regions are involved in the interaction with the host and may be locked in "arms race" coevolutionary conflicts.     

MSTF: a domain involved in bacterial metallopeptidases and surface proteins, mycobacteriophage tape-measure proteins and fungal proteins

Here we report a novel domain, MSTF (domain involved in bacterial metallopeptidases, surface proteins and other proteins, also present in mycobacteriophage tape-measure proteins and fungal proteins), which is present in bacteria, phages and fungi. MSTF is about 67-94 amino acids in length with one HxDHxH motif and some highly conserved residues including His, Gly, Ala and Asp. Secondary structure prediction indicated that this domain contains two alpha-helices and one beta-sheet. Identification of MSTF will provide an opportunity to develop new strategies to combat pathogenic microorganisms, especially Mycobacterium tuberculosis.     

A novel family of channel-forming,autotransporting, bacterial virulence factors

Pathogenic bacteria produce virulence factors that cross the bacterial cell envelope from the cytoplasm to the extracellular milieu where they promote disease. The mechanisms of their export are poorly understood. We here characterize a family of autotransporter (AT) protein domains present at the C-termini of several nonhomologous Gram-negative bacterial virulence factors. The family consist of 18 sequenced protein domains, the functionally characterized members of which catalyze export of (1) proteases, (2) virulence-related cell adhesins, (3) mediators of actin-promoted bacterial motility, (4) cytotoxins and (5) tissue invasion proteins. We (1) establish that these AT domains are homologous, (2) multiply align their sequences, (3) derive an AT family-specific signature sequence, and (4) define the evolutionary relationships between members of the family. Secondary structural predictions as well as average hydropathy, average similarity and average amphipathicity plots have allowed us to propose a specific 14 β-stranded barrel structural model that may be applicable to all protein members of the AT family. We suggest that the AT domains became associated with active virulence factor domains by interdomain fusion events that occurred during the evolution of these complex proteins.     

Organization of GPI-anchored proteins at the cell surface and its physiopathological relevance

Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are a class of proteins attached to the extracellular leaflet of the plasma membrane via a post-translational modification, the glycolipid anchor. The presence of both glycolipid anchor and protein portion confers them unique features. GPI-APs are expressed in all eukaryotes, from fungi to plants and animals. They display very diverse functions ranging from enzymatic activity, signaling, cell adhesion, cell wall metabolism, neuritogenesis, and immune response. Likewise other plasma membrane proteins, the spatio-temporal organization of GPI-APs is critical for their biological activities in physiological conditions. In this review, we will summarize the latest findings on plasma membrane organization of GPI-APs and the mechanism of its regulation in different cell types. We will also examine the involvement of specific GPI-APs namely the prion protein PrP^C, the Folate Receptor alpha and the urokinase plasminogen activator receptor in human diseases focusing on neurodegenerative diseases and cancer.     

On-fiber display of a functional peptide at sites distant from the cell surface using a long bacterionanofiber of a trimeric autotransporter adhesin

In the cell surface display system, the distance of a surface-displayed molecule from the cell surface should influence its functionality due to the interference by other surface structures. For the purpose of developing this distance-variable surface display system, we utilized a long fibrous adhesin, Acinetobacter trimeric autotransporter adhesin (AtaA) of the strain Tol 5. We constructed His-tagged full-length and shorter AtaA fibers designed by N-terminal deletion and expressed them in the ΔataA mutant. Immunoelectron microscopy clearly showed that they formed fibers on the cell surface and the His-tag was displayed on the fiber tip located at fixed distances from the cell surface. N-terminal deletion of AtaA shortened the distance between the His-tag and the cell surface, as designed. Time-course analyses of the cell-to-Ni-Sepharose beads binding revealed that cells producing the longer fibers bound more rapidly to the beads. The His-tagged AtaA derivatives were also displayed on Escherichia coli cells, and a similar tendency was shown; the His-tag on the longer fiber was more functional than that on the shorter one. Thus, we developed an on-fiber display system of a functional peptide using a long trimeric autotransporter adhesin (TAA) fiber, which can vary the distance between the displayed molecule and the cell surface.     

BamA forms a translocation channel for polypeptide export across the bacterial outer membrane

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Membranous structures transfer cell surface proteins across NK cell immune synapses

Intercellular transfer of cell surface proteins is widespread and facilitates several recently discovered means for immune cell communication. Here, we examined the molecular mechanism for intercellular exchange of the natural killer (NK) cell receptor KIR2DL1 and HLA-C, prototypical proteins that swap between NK cells and target cells. Transfer was contact dependent and enhanced for cells expressing cognate receptor/ligand pairs but did not depend on KIR2DL1 signaling. To a lesser extent, proteins transferred independent from specific recognition. Intracellular domains of transferred proteins were not exposed to the extracellular environment and transferred proteins were removed by brief exposure to low pH. By fluorescence microscopy, transferred proteins localized to discrete regions on the recipient cell surface. Higher resolution scanning electron micrographs revealed that transferred proteins were located within specific membranous structures. Transmission electron microscopy of the immune synapse revealed that membrane protrusions from one cell interacted with the apposing cell surface within the synaptic cleft. These data, coupled with previous observations, lead us to propose that intercellular protein transfer is mediated by membrane protrusions within and surrounding the immunological synapse.     

Crystal structure of the Haemophilus influenzae Hap adhesin reveals an intercellular oligomerization mechanism for bacterial aggregation

Bacterial biofilms are complex microbial communities that are common in nature and are being recognized increasingly as an important determinant of bacterial virulence. However, the structural determinants of bacterial aggregation and eventual biofilm formation have been poorly defined. In Gram‐negative bacteria, a major subgroup of extracellular proteins called self‐associating autotransporters (SAATs) can mediate cell–cell adhesion and facilitate biofilm formation. In this study, we used the Haemophilus influenzae Hap autotransporter as a prototype SAAT to understand how bacteria associate with each other. The crystal structure of the H. influenzae Hap_S passenger domain (harbouring the SAAT domain) was determined to 2.2 Å by X‐ray crystallography, revealing an unprecedented intercellular oligomerization mechanism for cell–cell interaction. The C‐terminal SAAT domain folds into a triangular‐prism‐like structure that can mediate Hap–Hap dimerization and higher degrees of multimerization through its F1–F2 edge and F2 face. The intercellular multimerization can give rise to massive buried surfaces that are required for overcoming the repulsive force between cells, leading to bacterial cell–cell interaction and formation of complex microcolonies.     

Polymorphic proteins of Chlamydia spp. – autotransporters beyond the Proteobacteria

Gram-negative bacteria secrete a variety of proteins to the cell surface and beyond, a process with many inherent difficulties. An exceptionally widespread answer to these problems is the type V (or autotransporter) secretion pathway. By exploiting the data made available by bacterial genome sequencing, we have discovered that the previously described polymorphic proteins of Chlamydia spp. resemble members of the autotransporter family, and we suggest that they follow the same secretion pathway.     

Dynamics of putative raft-associated proteins at the cell surface

Lipid rafts are conceptualized as membrane microdomains enriched in cholesterol and glycosphingolipid that serve as platforms for protein segregation and signaling. The properties of these domains in vivo are unclear. Here, we use fluorescence recovery after photobleaching to test if raft association affects a protein's ability to laterally diffuse large distances across the cell surface. The diffusion coefficients (D) of several types of putative raft and nonraft proteins were systematically measured under steady-state conditions and in response to raft perturbations. Raft proteins diffused freely over large distances (> 4 microm), exhibiting Ds that varied 10-fold. This finding indicates that raft proteins do not undergo long-range diffusion as part of discrete, stable raft domains. Perturbations reported to affect lipid rafts in model membrane systems or by biochemical fractionation (cholesterol depletion, decreased temperature, and cholesterol loading) had similar effects on the diffusional mobility of raft and nonraft proteins. Thus, raft association is not the dominant factor in determining long-range protein mobility at the cell surface.     

STIM1: a novel phosphoprotein located at the cell surface

STIM1 is a novel candidate growth suppressor gene mapping to the human chromosome region 11p15.5 that is associated with several malignancies. STIM1 overexpression studies in G401 rhabdoid tumour, rhabdomyosarcoma and rodent myoblast cell lines causes growth arrest, consistent with a potential role as a tumour growth suppressor. We used highly specific antibodies to show by immunofluorescence and cell surface biotinylation studies that STIM1 is located at the cell surface of K562 cells. Western blot analysis revealed that the 90-kDa STIM1 protein is ubiquitously expressed in various human primary cells and tumour cell lines. STIM1 is not secreted from cells and does not appear to undergo proteolytic processing. We show evidence of post-translational modification of STIM1, namely phosphorylation and N-linked glycosylation. Phosphorylation of STIM1 in vivo occurs predominantly on serine residues. Thus, STIM1, the putative tumour growth suppressor gene is ubiquitously expressed and has features of a regulatory cell-surface phosphoprotein.     

Detection and identification of bacterial cell surface proteins by fluorescent labeling

A rapid method of detection and identification of bacterial cell surface proteins is needed to better understand the interaction of bacteria with host components. To detect cell surface proteins, we have labeled cells of the Gram-negative anaerobic bacterium, Porphyromonas gingivalis, with fluorescent cyanine dyes, Cy3 and Cy5. We demonstrate that only cell surface proteins were labeled, indicating the method applied in our study is suitable for detection and identification of cell surface proteins in Gram-negative bacteria and possibly other organisms.     

Identification and analysis of novel tandem repeats in the cell surface proteins of archaeal and bacterial genomes using computational tools

We have identified four novel repeats and two domains in cell surface proteins encoded by the Methanosarcina acetivorans genome and in some archaeal and bacterial genomes. The repeats correspond to a certain number of amino acid residues present in tandem in a protein sequence and each repeat is characterized by conserved sequence motifs. These correspond to: (a) a 42 amino acid (aa) residue RIVW repeat; (b) a 45 aa residue LGxL repeat; (c) a 42 aa residue LVIVD repeat; and (d) a 54 aa residue LGFP repeat. The domains correspond to a certain number of aa residues in a protein sequence that do not comprise internal repeats. These correspond to: (a) a 200 aa residue DNRLRE domain; and (b) a 70 aa residue PEGA domain. We discuss the occurrence of these repeats and domains in the different proteins and genomes analysed in this work.     

Outer membrane protein PhoE as a carrier for the exposure of foreign antigenic determinants at the bacterial cell surface

PhoE protein is an abundant outer membrane protein of theEscherichia coli K-12 outer membrane. This protein can be used as an exposure system to produce foreign antigenic determinants and for their transport to the bacterial cell surface. The system is very flexible, since insertions varying in length and nature could be made in different cell surface-exposed regions of PhoE, without interfering with the assembly process of the mutant proteins into the outer membrane. Two antigenic determinants of the structural VP1 protein of foot-and-mouth disease virus were inserted in different combinations in four cell surface-exposed regions of PhoE. The epitopes were exposed at the bacterial cell surface and they keep their antigenic and immunogenic properties in this PhoE-associated conformation. Immunization of guinea pigs with one hybrid protein, containing a combination of the two epitopes inserted in the fourth exposed region, resulted in complete protection against challenge with the virus. A T-cell epitope of the 65 kDa heat shock protein ofMycobacterium tuberculosis was inserted in the fourth exposed region of PhoE and in vitro proliferation of two T-cell specific clones was demonstrated. Thus, the PhoE exposure system has been shown to be suitable for presentation of both B-cell and T-cell determinants to the immune system. Furthermore, good expression of the hybrid protein in attenuatedSalmonella strains, which can be used as live oral vaccines, was shown.Paper awarded the Kluyver Prize 1990 by the Netherlands Society of Microbiology to Dr. M.C. Agterberg     

Biotinylation and characterization of Cryptococcus neoformans cell surface proteins

AIMS: To develop a novel procedure for isolating and characterizing cryptococcal cell-surface proteins using biotinylation, fluorescein isothiocyanate (FITC)-streptavidin, flow cytometry and associated ligand-receptor analysis, confocal microscopy and electrophoretic separation. METHODS AND RESULTS: Cell proteins of both acapsulate and encapsulated Cryptococcus neoformans cells were labelled using sulfo-NHS-biotin which, in turn, was complexed with FITC-streptavidin. Resulting cell population fluorescence supported visualization of cell-surface protein distribution by confocal microscopy, as well as evaluation of protein exposure by flow cytometry and the calculation of the ligand-binding determinants EC(50), F(max) and H(n). Biotinylation of cell-surface proteins also supported their isolation by affinity chromatography and characterization by SDS/PAGE. Ligand-binding determinants, such as EC(50) values, indicated that acapsulate and stationary phase cells have greatest affinity for biotin. F(max) values demonstrated greatest protein exposure among stationary phase cells; in turn, encapsulated cells expose more protein than acapsulate counterparts. H(n) values of below unity potentially confirm the complex multi-receptor nature of biotin binding to cryptococcal cell surfaces under investigation. Fluorescence visualization showed marked but localized fluorescence indicative of protein exposure around sites of cell division. In turn, biotinylation of cell-surface proteins and their release under reducing conditions demonstrated at least two noncovalently linked proteinaceous entities, of 43 and 57 kDa, exposed on acapsulate cryptococcal cell walls. CONCLUSIONS: A novel method for identifying, in situ, cell-surface proteins exposed by C. neoformans was established. This novel technique was successfully implemented using both acapsulate and encapsulated C. neoformans cells, both were found to have dynamic and markedly localized protein distribution around sites of cell division and associated cell wall trauma. SIGNIFICANCE AND IMPACT OF THE STUDY: A novel procedure, employing a versatile combination of flow cytometry, ligand-receptor analysis, confocal microscopy and biotinylation, supported the characterization and isolation of cryptococcal cell-surface proteins.