Novel Bacterial Membrane Surface Display System Using Cell Wall-Less L-Forms of Proteus mirabilis and Escherichia coli

We describe a novel membrane surface display system that allows the anchoring of foreign proteins in the cytoplasmic membrane (CM) of stable, cell wall-less L-form cells of Escherichia coli and Proteus mirabilis. The reporter protein, staphylokinase (Sak), was fused to transmembrane domains of integral membrane proteins from E. coli (lactose permease LacY, preprotein translocase SecY) and P. mirabilis (curved cell morphology protein CcmA). Both L-form strains overexpressed fusion proteins in amounts of 1 to 100 μg ml⁻¹, with higher expression for those with homologous anchor motifs. Various experimental approaches, e.g., cell fractionation, Percoll gradient purification, and solubilization of the CM, demonstrated that the fusion proteins are tightly bound to the CM and do not form aggregates. Trypsin digestion, as well as electron microscopy of immunogold-labeled replicas, confirmed that the protein was localized on the outside surface. The displayed Sak showed functional activity, indicating correct folding. This membrane surface display system features endotoxin-poor organisms and can provide a novel platform for numerous applications.     

Display of Bacterial Lipase on the Escherichia coli Cell Surface by Using FadL as an Anchoring Motif and Use of the Enzyme in Enantioselective Biocatalysis

We have developed a novel cell surface display system by employing FadL as an anchoring motif, which is an outer membrane protein involved in long-chain fatty acid transport in Escherichia coli. A thermostable Bacillus sp. strain TG43 lipase (44.5 kDa) could be successfully displayed on the cell surface of E. coli in an active form by C-terminal deletion-fusion of lipase at the ninth external loop of FadL. The localization of the truncated FadL-lipase fusion protein on the cell surface was confirmed by confocal microscopy and Western blot analysis. Lipase activity was mainly detected with whole cells, but not with the culture supernatant, suggesting that cell lysis was not a problem. The activity of cell surface-displayed lipase was examined at different temperatures and pHs and was found to be the highest at 50°C and pH 9 to 10. Cell surface-displayed lipase was quite stable, even at 60 and 70°C, and retained over 90% of the full activity after incubation at 50°C for a week. As a potential application, cell surface-displayed lipase was used as a whole-cell catalyst for kinetic resolution of racemic methyl mandelate. In 36 h of reaction, (S)-mandelic acid could be produced with the enantiomeric excess of 99% and the enantiomeric ratio of 292, which are remarkably higher than values obtained with crude lipase or cross-linked lipase crystal. These results suggest that FadL may be a useful anchoring motif for displaying enzymes on the cell surface of E. coli for whole-cell biocatalysis.     

Study of Metal-Metal Interactions and Their Biomarkers Using an Intestinal Human Cell Line

Biological Trace Element Research - From the time of dietary intake to their utilization, the number of important interactions occurs among mineral elements, which can affect their bioavailability...     

Reconstitution and Engineering of Apoptotic Protein Interactions on the Bacterial Cell Surface

The interactions between pro- and anti-apoptotic members of the Bcl-2 class of proteins control whether a cell lives or dies, and the study of these protein-protein interactions has been an area of intense research. In this report, we describe a new tool for the study and engineering of apoptotic protein interactions that is based on the flow cytometric detection of these interactions on the surface of Escherichia coli. After validation of the assay with the well-studied interaction between the Bak(72-87) peptide and the anti-apoptotic protein Bcl-x_L, the effect of both increasing and decreasing Bak peptide length on Bcl-x_L binding was investigated. Previous work demonstrated that the Bak(72-87) peptide also binds to the anti-apoptotic protein Bcl-2, albeit with lower binding affinity compared to Bcl-x_L. Here, we demonstrate that a slightly longer Bak peptide corresponding to amino acids 72-89 of Bak binds Bcl-x_L and Bcl-2 equally well. Approximate binding affinity calculations on these peptide-protein complexes confirm the experimental observations. The flow cytometric assay was also used to screen a saturation mutagenesis library of Bak(72-87) variants for improved affinity to Bcl-x_L. The best variants obtained from this library exhibit an apparent K_d to Bcl-x_L 4-fold lower than that of wild-type Bak(72-87).     

Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority of CRC cases, cell dysfunction is the result of numerous mutations that modify protein expression and post-translational modification¹.A number of cell surface antigens, including cluster of differentiation (CD) antigens, have been identified as potential prognostic or metastatic biomarkers in CRC. These antigens make ideal biomarkers as their expression often changes with tumour progression or interactions with other cell types, such as tumour-infiltrating lymphocytes (TILs) and tumour-associated macrophages (TAMs).The use of immunohistochemistry (IHC) for cancer sub-classification and prognostication is well established for some tumour types^2,3. However, no single ‘marker’ has shown prognostic significance greater than clinico-pathological staging or gained wide acceptance for use in routine pathology reporting of all CRC cases. A more recent approach to prognostic stratification of disease phenotypes relies on surface protein profiles using multiple ''markers''. While expression profiling of tumours using proteomic techniques such as iTRAQ is a powerful tool for the discovery of biomarkers4, it is not optimal for routine use in diagnostic laboratories and cannot distinguish different cell types in a mixed population. In addition, large amounts of tumour tissue are required for the profiling of purified plasma membrane glycoproteins by these methods. In this video we described a simple method for surface proteome profiling of viable cells from disaggregated CRC samples using a DotScan CRC antibody microarray. The 122-antibody microarray consists of a standard 82-antibody region recognizing a range of lineage-specific leukocyte markers, adhesion molecules, receptors and markers of inflammation and immune response⁵, together with a satellite region for detection of 40 potentially prognostic markers for CRC. Cells are captured only on antibodies for which they express the corresponding antigen. The cell density per dot, determined by optical scanning, reflects the proportion of cells expressing that antigen, the level of expression of the antigen and affinity of the antibody⁶. For CRC tissue or normal intestinal mucosa, optical scans reflect the immunophenotype of mixed populations of cells. Fluorescence multiplexing can then be used to profile selected sub-populations of cells of interest captured on the array. For example, Alexa 647-anti-epithelial cell adhesion molecule (EpCAM; CD326), is a pan-epithelial differentiation antigen that was used to detect CRC cells and also epithelial cells of normal intestinal mucosa, while Phycoerythrin-anti-CD3, was used to detect infiltrating T-cells⁷. The DotScan CRC microarray should be the prototype for a diagnostic alternative to the anatomically-based CRC staging system.     

Surface Proteins of Gram-Positive Bacteria and Mechanisms of Their Targeting to the Cell Wall Envelope

The cell wall envelope of gram-positive bacteria is a macromolecular, exoskeletal organelle that is assembled and turned over at designated sites. The cell wall also functions as a surface organelle that allows gram-positive pathogens to interact with their environment, in particular the tissues of the infected host. All of these functions require that surface proteins and enzymes be properly targeted to the cell wall envelope. Two basic mechanisms, cell wall sorting and targeting, have been identified. Cell well sorting is the covalent attachment of surface proteins to the peptidoglycan via a C-terminal sorting signal that contains a consensus LPXTG sequence. More than 100 proteins that possess cell wall-sorting signals, including the M proteins of Streptococcus pyogenes, protein A of Staphylococcus aureus, and several internalins of Listeria monocytogenes, have been identified. Cell wall targeting involves the noncovalent attachment of proteins to the cell surface via specialized binding domains. Several of these wall-binding domains appear to interact with secondary wall polymers that are associated with the peptidoglycan, for example teichoic acids and polysaccharides. Proteins that are targeted to the cell surface include muralytic enzymes such as autolysins, lysostaphin, and phage lytic enzymes. Other examples for targeted proteins are the surface S-layer proteins of bacilli and clostridia, as well as virulence factors required for the pathogenesis of L. monocytogenes (internalin B) and Streptococcus pneumoniae (PspA) infections. In this review we describe the mechanisms for both sorting and targeting of proteins to the envelope of gram-positive bacteria and review the functions of known surface proteins.     

Investigation of the Cell Surface Structures of Agrobacteria Involved in Bacterial and Plant Interactions

Agrobacterial cells produced straight microfibrils not only when in contact with wheat seedling roots, but also when in contact with each other. After 2 h of incubation, agrobacterial cells were found to form aggregates, in which the cells were in contact either directly or through thick straight microfibrils (bridges) of an unknown composition. The majority of the microfibrils were susceptible to attack by cellulase, although some of them showed resistance to this enzyme. Like the wild-type flagellated agrobacteria, their bald mutants produced long straight microfibrils. The cell surface structures of agrobacteria were examined by labeling them immunocytochemically with colloidal gold–conjugated antibodies against O-specific lipopolysaccharides, Vir proteins, and cellulase. Agrobacterial cells treated with acetosyringone and brought into contact were found to contain subpolar and polar cell surface structures. Antibodies against the VirB2 protein were able to interact with a tuft of thin microfibrils located on one pole of the agrobacterial cell whose virgenes were induced by acetosyringone but were unable to interact with the surface structures of the agrobacterial cells aggregated in liquid medium in the absence of wheat seedlings.     

Screening for Glycosylphosphatidylinositol-Modified Cell Wall Proteins in Pichia pastoris and Their Recombinant Expression on the Cell Surface

Glycosylphosphatidylinositol (GPI)-anchored glycoproteins have various intrinsic functions in yeasts and different uses in vitro. In the present study, the genome of Pichia pastoris GS115 was screened for potential GPI-modified cell wall proteins. Fifty putative GPI-anchored proteins were selected on the basis of (i) the presence of a C-terminal GPI attachment signal sequence, (ii) the presence of an N-terminal signal sequence for secretion, and (iii) the absence of transmembrane domains in mature protein. The predicted GPI-anchored proteins were fused to an alpha-factor secretion signal as a substitute for their own N-terminal signal peptides and tagged with the chimeric reporters FLAG tag and mature Candida antarctica lipase B (CALB). The expression of fusion proteins on the cell surface of P. pastoris GS115 was determined by whole-cell flow cytometry and immunoblotting analysis of the cell wall extracts obtained by β-1,3-glucanase digestion. CALB displayed on the cell surface of P. pastoris GS115 with the predicted GPI-anchored proteins was examined on the basis of potential hydrolysis of p-nitrophenyl butyrate. Finally, 13 proteins were confirmed to be GPI-modified cell wall proteins in P. pastoris GS115, which can be used to display heterologous proteins on the yeast cell surface.     

Export from Pericentriolar Endocytic Recycling Compartment to Cell Surface Depends on Stable, Detyrosinated (Glu) Microtubules and Kinesin

A significant fraction of internalized transferrin (Tf) concentrates in the endocytic recycling compartment (ERC), which is near the microtubule-organizing center in many cell types. Tf then recycles back to the cell surface. The mechanisms controlling the localization, morphology, and function of the ERC are not fully understood. We examined the relationship of Tf trafficking with microtubules (MTs), specifically the subset of stable, detyrosinated Glu MTs. We found some correlation between the level of stable Glu MTs and the distribution of the ERC; in cells with low levels of Glu MTs concentrated near to the centriole, the ERC was often tightly clustered, whereas in cells with higher levels of Glu MTs throughout the cell, the ERC was more dispersed. The clustered ERC in Chinese hamster ovary cells became dispersed when the level of Glu MTs was increased with taxol treatment. Furthermore, in a temperature-sensitive Chinese hamster ovary cell line (B104-5), the cells had more Glu MTs when the ERC became dispersed at elevated temperature. Microinjecting purified anti-Glu tubulin antibody into B104-5 cells at elevated temperature induced the redistribution of the ERC to a tight cluster. Microinjection of anti-Glu tubulin antibody slowed recycling of Tf to the cell surface without affecting Tf internalization or delivery to the ERC. Similar inhibition of Tf recycling was caused by microinjecting anti-kinesin antibody. These results suggest that stable Glu MTs and kinesin play a role in the organization of the ERC and in facilitating movement of vesicles from the ERC to the cell surface.     

家蝇抗菌肽对细菌细胞表面特性影响及其作用机理的研究

利用微生物对十六烷吸附的方法(MATS方法)、微电泳方法与测定细菌质膜上β-半乳糖苷酶活性的方法,探讨了家蝇抗菌肽对大肠杆菌等6种细菌细胞表面特性及其细胞膜的作用机制。研究结果表明,抗菌肽使细菌表面电负性增强,对G 细菌细胞表面电荷的改变大于对G-的改变,使细菌细胞表面疏水性不同程度的下降。抗菌肽引起细菌细胞膜通透性迅速增加,不同细菌β-半乳糖苷酶释放的最大速度VP在3.86pmol/min～6.92pmol/min,相应的时间TP为0,由此推测抗菌肽对细胞膜的作用机制是“形成孔洞”。     

Re-Directing an Alkylating Agent to Mitochondria Alters Drug Target and Cell Death Mechanism

We have successfully delivered a reactive alkylating agent, chlorambucil (Cbl), to the mitochondria of mammalian cells. Here, we characterize the mechanism of cell death for mitochondria-targeted chlorambucil (mt-Cbl) in vitro and assess its efficacy in a xenograft mouse model of leukemia. Using a ρ° cell model, we show that mt-Cbl toxicity is not dependent on mitochondrial DNA damage. We also illustrate that re-targeting Cbl to mitochondria results in a shift in the cell death mechanism from apoptosis to necrosis, and that this behavior is a general feature of mitochondria-targeted Cbl. Despite the change in cell death mechanisms, we show that mt-Cbl is still effective in vivo and has an improved pharmacokinetic profile compared to the parent drug. These findings illustrate that mitochondrial rerouting changes the site of action of Cbl and also alters the cell death mechanism drastically without compromising in vivo efficacy. Thus, mitochondrial delivery allows the exploitation of Cbl as a promiscuous mitochondrial protein inhibitor with promising therapeutic potential.     

Amphibian Lens Histones and Their Relation to the Cell Cycle

Histones have been electrophoretically separated from acid extracts of the frog lens for the first time. The five conventional histone fractions, representing four electrophoretic bands (f1; f2b, f3; f2a2; and f2a1), are present in both the epithelial and fiber cells. In addition, a fifth fraction was isolated from both sources and the evidence suggests that it may be a tissue-specific histone, possibly related to the lysine-rich f2c fraction found previously only in nucleated erythrocytes. The epithelial cells contain a substantially greater amount of histone than the fiber cells. Moreover, the fibers, unlike the epithelium, manifest no net histone synthesis or turnover following lenticular explantation. Microspectrophotometric, radioautographic, and gel electrophoretic studies indicate that the histones are synthesized in frog lenses concurrently with DNA. Inhibition of DNA synthesis does not completely abolish that of histones but reduces it by about one-half. In the early stages of culture (prior to their synthesis and that of DNA) the histones appear to undergo alterations which are prevented by treatment with cycloheximide.     

Human Herpesvirus 7 U21 Downregulates Classical and Nonclassical Class I Major Histocompatibility Complex Molecules from the Cell Surface

Herpesviruses have evolved numerous strategies to evade detection by the immune system. Notably, most of the herpesviruses interfere with viral antigen presentation to cytotoxic T lymphocytes (CTLs) by removing class I major histocompatibility complex (MHC) molecules from the infected cell surface. Clearly, since the herpesviruses have evolved an extensive array of mechanisms to remove class I MHC molecules from the cell surface, this strategy serves them well. However, class I MHC molecules often serve as inhibitory ligands for NK cells, so viral downregulation of all class I MHC molecules should leave the infected cell open to NK cell attack. Some viruses solve this problem by selectively downregulating certain class I MHC products, leaving other class I products at the cell surface to serve as inhibitory NK cell ligands. Here, we show that human herpesvirus 7 (HHV-7) U21 binds to and downregulates all of the human class I MHC gene products, as well as the murine class I molecule H-2K^b. HHV-7-infected cells must therefore possess other means of escaping NK cell detection.Human herpesvirus-7 (HHV-7) is a betaherpesvirus that infects over 90% of the population by the age of 3 (for a review, see reference 58). Like all other herpesviruses, HHV-7 establishes a latent or persistent infection, lasting for the lifetime of its host. Primary infection is usually accompanied by febrile illness, but long-term infection with the virus is asymptomatic (3, 53). HHV-7 is T-lymphotrophic, but it has also been found in salivary epithelial cells (30, 62).As viruses that remain latent or persistent throughout the life of their hosts, the herpesviruses must interact continually with the host immune system. In so doing, all herpesviruses have evolved mechanisms to interfere with viral antigen presentation by class I major histocompatibility complex (MHC) molecules as a means to escape detection by cytotoxic T lymphocytes (CTLs). Some herpesvirus gene products interfere with proteolysis of antigens or peptide transport into the endoplasmic reticulum (ER) (1, 20, 56, 61). Others retain or destroy class I molecules (2, 26, 59, 64), enhance the internalization of class I molecules, or divert class I molecules to lysosomes for degradation (11, 23, 25, 44). Judging from the number and molecular diversity of these strategies, the removal of MHC class I-peptide complexes from the cell surface must be evolutionarily advantageous to these viruses as a means of escaping immune detection. We have described one such immunoevasin, U21, from HHV-7. HHV-7 U21 binds to class I MHC molecules in the ER and diverts them to a lysosomal compartment, where they are degraded, effectively removing them from the cell surface (23). The mechanism of U21-mediated diversion of class I molecules to lysosomes is not known, but the relocalization of class I MHC molecules is specific—U21 does not cause the rerouting of either the transferrin receptor or CD4 to lysosomes (22, 23).Since the herpesviruses have evolved such an extensive array of mechanisms to remove class I MHC molecules from the cell surface of infected cells, this strategy must serve them well. However, when natural killer (NK) cells detect an absence of class I MHC molecules on the surface of a cell (i.e., “missing self”), they become activated to kill that cell. NK cells detect the absence of class I MHC molecules through interaction of NK cell receptors with NK cell receptor ligands present on the surface of the target cell (for a review, see references 6 and 7). When an NK cell surveys a potential target, it integrates the number and strength of the activating and inhibitory signals it receives; after weighing the balance, it either remains indifferent to the target or becomes activated to kill it.Class I MHC molecules are ligands for inhibitory NK cell receptors. Thus, when a virus removes class I MHC molecules from the cell surface to escape detection by CTLs, it simultaneously renders the cell vulnerable to NK cell attack. Not surprisingly, viruses have evolved counterstrategies to protect their host cells from NK cell-mediated attack. The class I MHC locus contains three classical class I gene products, HLA-A, -B, and -C, as well as other “nonclassical” products, including HLA-E and HLA-G. As a strategy to avoid both CTL and NK cell attack, some viral immunoevasins selectively downregulate HLA-A and HLA-B locus products, while leaving HLA-C, -E, and other inhibitory class I-like molecules at the plasma membrane (10, 16, 35). It has therefore been speculated that HLA-A and -B may be more effective at antigen presentation to CTLs than HLA-C (15, 40). The nonclassical class I molecule HLA-E, on the other hand, functions primarily to inhibit NK cell activation and does not present foreign antigen to CTLs (33). As such, its expression at the cell surface is even promoted by at least one immunoevasin, UL40 from human cytomegalovirus (HCMV) (54, 57).We do not know how HHV-7 responds to the selective pressures exerted by NK cells. We have shown previously that U21 can associate with and downregulate HLA-A and -B, but we do not yet know the full extent of its promiscuity (23). For this reason, we now examine the ability of U21 to bind to and downregulate the various classical and nonclassical class I MHC gene products. We find that, unlike many other viral immunoevasins, HHV-7 U21 can associate with and downregulate HLA-C, -E, and -G and even murine class I MHC molecules. In an infection, this would shift the balance of inhibitory NK cell ligands on the cell surface to favor NK cell attack, suggesting that HHV-7 might compensate for such an imbalance through other means of NK cell evasion.U21 is 55-kDa type I membrane protein with a short (50-amino-acid [aa]) cytoplasmic tail. We have shown that its transmembrane domain and cytoplasmic tail are not involved in its association with the lumenal domain of the class I molecule (22). In addition to gaining information about U21''s potential influence on CTL and NK cell detection of HHV-7-infected cells, we also hoped that a survey of its ability to associate with various class I MHC gene products might help to illuminate regions of the class I molecule important for association with U21.     

Correlation Between the Expression of an Escherichia coli Cell Surface Protein and the Ability of the Protein to Bind to Lipopolysaccharide

The ompA gene of Escherichia coli codes for a major protein of the outer membrane. When this gene was moved between various unrelated strains (E. coli K-12 and two clinical isolates of E. coli) by transduction, the gene was expressed very poorly. Recombinants carrying “foreign” genes produced no OmpA protein which could be detected on polyacrylamide gels and became resistant to bacteriophage K3, which uses this protein as receptor. The recombinants were sensitive to host-range mutants of K3, indicating a very low level of OmpA protein was produced. When an E. coli K-12 recombinant carrying an unexpressed foreign ompA allele was subjected to two cycles of selection for an OmpA⁺ phenotype, a mutant strain was obtained which was sensitive to K3 and which expressed nearly normal levels of OmpA protein in the outer membrane. This strain carried mutations in the foreign ompA gene, as indicated both by genetic mapping and the alteration of a peptide in the mutant OmpA protein. The ability of the OmpA protein to bind to lipopolysaccharide (LPS) showed similar strain specificity, and the mutant OmpA protein which was expressed in an unrelated host showed enhanced ability to bind LPS from its new host. Thus, cell surface expression of the ompA gene appears to depend upon the ability of the gene product to bind LPS, suggesting that an interaction between the protein and LPS plays an essential role in biosynthesis of this outer membrane protein.     

Display of Polyhistidine Peptides on the Escherichia coli Cell Surface by Using Outer Membrane Protein C as an Anchoring Motif

A novel cell surface display system was developed by employing Escherichia coli outer membrane protein C (OmpC) as an anchoring motif. Polyhistidine peptides consisting of up to 162 amino acids could be successfully displayed on the seventh exposed loop of OmpC. Recombinant cells displaying polyhistidine could adsorb up to 32.0 μmol of Cd²⁺ per g (dry weight) of cells.     

Mpl Traffics to the Cell Surface Through Conventional and Unconventional Routes

Myeloproliferative neoplasms (MPNs) are often characterized by JAK2 or calreticulin (CALR) mutations, indicating aberrant trafficking in pathogenesis. This study focuses on Mpl trafficking and Jak2 association using two model systems: human erythroleukemia cells (HEL; JAK2V617F) and K562 myeloid leukemia cells (JAK2WT). Consistent with a putative chaperone role for Jak2, Mpl and Jak2 associate on both intracellular and plasma membranes (shown by proximity ligation assay) and siRNA‐mediated knockdown of Jak2 led to Mpl trapping in the endoplasmic reticulum (ER). Even in Jak2 sufficient cells, Mpl accumulates in punctate structures that partially colocalize with ER‐tracker, the ER exit site marker (ERES) Sec31a, the autophagy marker LC3 and LAMP1. Mpl was fused to miniSOG, a genetically encoded tag for correlated light and electron microscopy. Results suggest that a fraction of Mpl is taken up into autophagic structures from the ER and routed to autolyososomes. Surface biotinylation shows that both immature and mature Mpl reach the cell surface; in K562 cells Mpl is also released in exosomes. Both forms rapidly internalize upon ligand addition, while recovery is primarily attributed to immature Mpl. Mpl appears to reach the plasma membrane via both conventional ER‐Golgi and autolysosome secretory pathways, as well as recycling.      

Pathogenic Acinetobacter: from the Cell Surface to Infinity and Beyond

The genus Acinetobacter encompasses multiple nosocomial opportunistic pathogens that are of increasing worldwide relevance because of their ability to survive exposure to various antimicrobial and sterilization agents. Among these, Acinetobacter baumannii, Acinetobacter nosocomialis, and Acinetobacter pittii are the most frequently isolated in hospitals around the world. Despite the growing incidence of multidrug-resistant Acinetobacter spp., little is known about the factors that contribute to pathogenesis. New strategies for treating and managing infections caused by multidrug-resistant Acinetobacter strains are urgently needed, and this requires a detailed understanding of the pathobiology of these organisms. In recent years, some virulence factors important for Acinetobacter colonization have started to emerge. In this review, we focus on several recently described virulence factors that act at the bacterial surface level, such as the capsule, O-linked protein glycosylation, and adhesins. Furthermore, we describe the current knowledge regarding the type II and type VI secretion systems present in these strains.