Bacterial Macroscopic Rope-like Fibers with Cytopathic and Adhesive Properties

We present a body of ultrastructural, biochemical, and genetic evidence that demonstrates the oligomerization of virulence-associated autotransporter proteins EspC or EspP produced by deadly human pathogens enterohemorrhagic and enteropathogenic Escherichia coli into novel macroscopic rope-like structures (>1 cm long). The rope-like structures showed high aggregation and insolubility, stability to anionic detergents and high temperature, and binding to Congo Red and thioflavin T dyes. These are properties also exhibited by human amyloidogenic proteins. These macroscopic ropes were not observed in cultures of nonpathogenic Escherichia coli or isogenic espP or espC deletion mutants of enterohemorrhagic or enteropathogenic Escherichia coli but were produced by an Escherichia coli K-12 strain carrying a plasmid expressing espP. Purified recombinant EspP monomers were able to self-assemble into macroscopic ropes upon incubation, suggesting that no other protein was required for assembly. The ropes bound to and showed cytopathic effects on cultured epithelial cells, served as a substratum for bacterial adherence and biofilm formation, and protected bacteria from antimicrobial compounds. We hypothesize that these ropes play a biologically significant role in the survival and pathogenic scheme of these organisms.     

Crystal structure of the passenger domain of the Escherichia coli autotransporter EspP

Autotransporters represent a large superfamily of known and putative virulence factors produced by Gram-negative bacteria. They consist of an N-terminal “passenger domain” responsible for the specific effector functions of the molecule and a C-terminal “β-domain” responsible for translocation of the passenger across the bacterial outer membrane. Here, we present the 2.5-Å crystal structure of the passenger domain of the extracellular serine protease EspP, produced by the pathogen Escherichia coli O157:H7 and a member of the serine protease autotransporters of Enterobacteriaceae (SPATEs). Like the previously structurally characterized SPATE passenger domains, the EspP passenger domain contains an extended right-handed parallel β-helix preceded by an N-terminal globular domain housing the catalytic function of the protease. Of note, however, is the absence of a second globular domain protruding from this β-helix. We describe the structure of the EspP passenger domain in the context of previous results and provide an alternative hypothesis for the function of the β-helix within SPATEs.     

Autotransporter-Based Antigen Display in Bacterial Ghosts

Bacterial ghosts are empty cell envelopes of Gram-negative bacteria that can be used as vehicles for antigen delivery. Ghosts are generated by releasing the bacterial cytoplasmic contents through a channel in the cell envelope that is created by the controlled production of the bacteriophage ϕX174 lysis protein E. While ghosts possess all the immunostimulatory surface properties of the original host strain, they do not pose any of the infectious threats associated with live vaccines. Recently, we have engineered the Escherichia coli autotransporter hemoglobin protease (Hbp) into a platform for the efficient surface display of heterologous proteins in Gram-negative bacteria, HbpD. Using the Mycobacterium tuberculosis vaccine target ESAT6 (early secreted antigenic target of 6 kDa), we have explored the application of HbpD to decorate E. coli and Salmonella ghosts with antigens. The use of different promoter systems enabled the concerted production of HbpD-ESAT6 and lysis protein E. Ghost formation was monitored by determining lysis efficiency based on CFU, the localization of a set of cellular markers, fluorescence microscopy, flow cytometry, and electron microscopy. Hbp-mediated surface display of ESAT6 was monitored using a combination of a protease accessibility assay, fluorescence microscopy, flow cytometry and (immuno-)electron microscopy. Here, we show that the concerted production of HbpD and lysis protein E in E. coli and Salmonella can be used to produce ghosts that efficiently display antigens on their surface. This system holds promise for the development of safe and cost-effective vaccines with optimal intrinsic adjuvant activity and exposure of heterologous antigens to the immune system.     

Use of Pseudomonas putida EstA as an Anchoring Motif for Display of a Periplasmic Enzyme on the Surface of Escherichia coli

The functional expression of proteins on the surface of bacteria has proven important for numerous biotechnological applications. In this report, we investigated the N-terminal fusion display of the periplasmic enzyme β-lactamase (Bla) on the surface of Escherichia coli by using the translocator domain of the Pseudomonas putida outer membrane esterase (EstA), which is a member of the lipolytic autotransporter enzymes. To find out the transport function of a C-terminal domain of EstA, we generated a set of Bla-EstA fusion proteins containing N-terminally truncated derivatives of the EstA C-terminal domain. The surface exposure of the Bla moiety was verified by whole-cell immunoblots, protease accessibility, and fluorescence-activated cell sorting. The investigation of growth kinetics and host cell viability showed that the presence of the EstA translocator domain in the outer membrane neither inhibits cell growth nor affects cell viability. Furthermore, the surface-exposed Bla moiety was shown to be enzymatically active. These results demonstrate for the first time that the translocator domain of a lipolytic autotransporter enzyme is an effective anchoring motif for the functional display of heterologous passenger protein on the surface of E. coli. This investigation also provides a possible topological model of the EstA translocator domain, which might serve as a basis for the construction of fusion proteins containing heterologous passenger domains.     

X-ray crystal structure of the passenger domain of plasmid encoded toxin(Pet), an autotransporter enterotoxin from enteroaggregative Escherichia coli (EAEC)

Autotransporters (ATs) represent a superfamily of proteins produced by a variety of pathogenic bacteria, which include the pathogenic groups of Escherichia coli (E. coli) associated with gastrointestinal and urinary tract infections. We present the first X-ray structure of the passenger domain from the Plasmid-encoded toxin (Pet) a 100 kDa protein at 2.3 Å resolution which is a cause of acute diarrhea in both developing and industrialized countries. Pet is a cytoskeleton-altering toxin that induces loss of actin stress fibers. While Pet (pdb code: 4OM9) shows only a sequence identity of 50% compared to the closest related protein sequence, extracellular serine protease plasmid (EspP) the structural features of both proteins are conserved. A closer structural look reveals that Pet contains a β-pleaded sheet at the sequence region of residues 181–190, the corresponding structural domain in EspP consists of a coiled loop. Secondary, the Pet passenger domain features a more pronounced beta sheet between residues 135 and 143 compared to the structure of EspP.     

Sp1 modulates ncOGT activity to alter target recognition and enhanced thermotolerance in E. coli

cDNAs encoding three isoforms of OGT (ncOGT, mOGT, and sOGT) were expressed in Escherichia coli in which the coexpression system of OGT with target substrates was established in vivo. No endogenous bacterial proteins were significantly O-GlcNAcylated by any type of OGT isoform while co-expressed p62 and Sp1 were strongly O-GlcNAcylated by ncOGT. These results suggest that most of bacterial proteins appear not to be recognized as right substrates by mammalian OGT whereas cytosolic environments may supply UDP-GlcNAc enough to proceed to O-GlcNAcylation in E. coli. Under these conditions, sOGT was auto-O-GlcNAcylated whereas ncOGT and mOGT were not. Importantly, we found that when Sp1 was coexpressed, ncOGT can O-GlcNAcylate not only Sp1 but also many bacterial proteins. Our findings suggest that Sp1 may modulate the capability of target recognition of ncOGT by which ncOGT can be led to newly recognize bacterial proteins as target substrates, finally generating the O-glyco-bacteria. Our results demonstrate that the O-glyco-bacteria showed enhanced thermal resistance to allow cell survival at a temperature as high as 52 °C.     

The trimeric periplasmic chaperone Skp of Escherichia coli forms 1:1 complexes with outer membrane proteins via hydrophobic and electrostatic interactions

The interactions of outer membrane proteins (OMPs) with the periplasmic chaperone Skp from Escherichia coli are not well understood. We have examined the binding of Skp to various OMPs of different origin, size, and function. These were OmpA, OmpG, and YaeT (Omp85) from Escherichia coli, the translocator domain of the autotransporter NalP from Neisseria meningitides, FomA from Fusobacterium nucleatum, and the voltage-dependent anion-selective channel, human isoform 1 (hVDAC1) from mitochondria. Binding of Skp was observed for bacterial OMPs, but neither for hVDAC1 nor for soluble bovine serum albumin. The Skp trimer formed 1:1 complexes, OMP·Skp₃, with bacterial OMPs, independent of their size or origin. The dissociation constants of these OMP·Skp₃ complexes were all in the nanomolar range, indicating that they are stable. Complexes of Skp₃ with YaeT displayed the smallest dissociation constants, complexes with NalP the largest. OMP binding to Skp₃ was pH-dependent and not observed when either Skp or OMPs were neutralized at very basic or very acidic pH. When the ionic strength was increased, the free energies of binding of Skp to OmpA or OmpG were reduced. Electrostatic interactions were therefore necessary for formation and stability of OMP·Skp₃ complexes. Light-scattering and circular dichroism experiments demonstrated that Skp₃ remained a stable trimer from pH 3 to pH 11. In the OmpA·Skp₃ complex, Skp efficiently shielded tryptophan residues of the transmembrane strands of OmpA against fluorescence quenching by aqueous acrylamide. Lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, bound to OmpA·Skp₃ complexes at low stoichiometries. Acrylamide quenching of fluorescence indicated that in this ternary complex, the tryptophan residues of the transmembrane domain of OmpA were located closer to the surface than in binary OmpA·Skp₃ complexes. This may explain previous observations that folding of Skp-bound OmpA into lipid bilayers is facilitated in presence of LPS.     

Structure and Function Relationship of the Autotransport and Proteolytic Activity of EspP from Shiga Toxin-Producing Escherichia coli

Background

The serine protease autotransporter EspP is a proposed virulence factor of Shiga toxin-producing Escherichia coli (STEC). We recently distinguished four EspP subtypes (EspPα, EspPβ, EspPγ, and EspPδ), which display large differences in transport and proteolytic activities and differ widely concerning their distribution within the STEC population. The mechanisms underlying these functional variations in EspP subtypes are, however, unknown.

Methodology/Principal Findings

The structural basis of proteolytic and autotransport activity was investigated using transposon-based linker scanning mutagenesis, site-directed mutagenesis and structure-function analysis derived from homology modelling of the EspP passenger domain. Transposon mutagenesis of the passenger domain inactivated autotransport when pentapeptide linker insertions occurred in regions essential for overall correct folding or in a loop protruding from the β-helical core. Loss of proteolytic function was limited to mutations in Domain 1 in the N-terminal third of the EspP passenger. Site-directed mutagenesis demonstrated that His¹²⁷, Asp¹⁵⁶ and Ser²⁶³ in Domain 1 form the catalytic triad of EspP.

Conclusions/Significance

Our data indicate that in EspP i) the correct formation of the tertiary structure of the passenger domain is essential for efficient autotransport, and ii) an elastase-like serine protease domain in the N-terminal Domain 1 is responsible for the proteolytic phenotype. Lack of stabilizing interactions of Domain 1 with the core structure of the passenger domain ablates proteolytic activity in subtypes EspPβ and EspPδ.     

Molecular Basis for the Structural Stability of an Enclosed β-Barrel Loop

We present molecular dynamics simulation studies of the structural stability of an enclosed loop in the β domain of the Escherichia coli O157:H7 autotransporter EspP. Our investigation revealed that, in addition to its excellent resistance to thermal perturbations, EspP loop 5 (L5) also has remarkable mechanical stability against pulling forces along the membrane norm. These findings are consistent with the experimental report that EspP L5 helps to maintain the permeability barrier in the outer membrane. In contrast to the major secondary structure elements of globular proteins such as ubiquitin, whose resistance to thermal and mechanical perturbations depends mainly on backbone hydrogen bonds and hydrophobic interactions, the structural stability of EspP L5 can be attributed mainly to geometric constraints and side-chain interactions dominated by hydrogen bonds. Examination of B-factors from available high-resolution structures of membrane-embedded β barrels indicates that most of the enclosed loops have stable structures. This finding suggests that loops stabilized by geometric constraints and side-chain interactions might be used more generally to restrict β-barrel channels for various functional purposes.     

Stepwise Folding of an Autotransporter Passenger Domain Is Not Essential for Its Secretion

Autotransporters are a superfamily of virulence proteins produced by Gram-negative bacteria. They consist of an N-terminal β-helical domain (“passenger domain”) that is secreted into the extracellular space and a C-terminal β-barrel domain (“β-domain”) that anchors the protein to the outer membrane. Because the periplasm lacks ATP, vectorial folding of the passenger domain in a C-to-N-terminal direction has been proposed to drive the secretion reaction. Consistent with this hypothesis, mutations that disrupt the folding of the C terminus of the passenger domain of the Escherichia coli O157:H7 autotransporter EspP have been shown to cause strong secretion defects. Here, we show that point mutations introduced at specific locations near the middle or N terminus of the EspP β-helix that perturb folding also impair secretion, but to a lesser degree. Surprisingly, we found that even multiple mutations that potentially abolish the stability of several consecutive rungs of the β-helix only moderately reduce secretion efficiency. Although these results provide evidence that the free energy derived from passenger domain folding contributes to secretion efficiency, they also suggest that a significant fraction of the energy required for secretion is derived from another source.     

Structure-guided engineering of Anticalins with improved binding behavior and biochemical characteristics for application in radio-immuno imaging and/or therapy

Modern strategies in radio-immuno therapy and in vivo imaging require robust, small, and specific ligand-binding proteins. In this context we have previously developed artificial lipocalins, so-called Anticalins, with high binding activity toward rare-earth metal–chelate complexes using combinatorial protein design. Here we describe further improvement of the Anticalin C26 via in vitro affinity maturation to yield CL31, which has a fourfold slower dissociation half-life above 2 h. Also, we present the crystallographic analyses of both the initial and the improved Anticalin, providing insight into the molecular mechanism of chelated metal binding and the role of amino acid substitutions during the step-wise affinity maturation. Notably, one of the four structurally variable loops that form the ligand pocket in the lipocalin scaffold undergoes a significant conformational change from C26 to CL31, acting as a lid that closes over the accommodated metal–chelate ligand. A systematic mutational study indicated that further improvement of ligand affinity is difficult to achieve while providing clues on the contribution of relevant side chains in the engineered binding pocket. Unexpectedly, some of the amino acid replacements led to strong increases – more then 10-fold – in the yield of soluble protein from periplasmic secretion in Escherichia coli.     

Subtypes of the Plasmid-Encoded Serine Protease EspP in Shiga Toxin-Producing Escherichia coli: Distribution, Secretion, and Proteolytic Activity

We investigated the prevalence, distribution, and structure of espP in Shiga toxin-producing Escherichia coli (STEC) and assessed the secretion and proteolytic activity of the encoded autotransporter protein EspP (extracellular serine protease, plasmid encoded). espP was identified in 56 of 107 different STEC serotypes. Sequencing of a 3,747-bp region of the 3,900-bp espP gene distinguished four alleles (espPα, espPβ, espPγ, and espPδ), with 99.9%, 99.2%, 95.3%, and 95.1% homology, respectively, to espP of E. coli O157:H7 strain EDL933. The espPβ, espPγ, and espPδ genes contained unique insertions and/or clustered point mutations that enabled allele-specific PCRs; these demonstrated the presence of espPα, espPβ, espPγ, and espPδ in STEC isolates belonging to 17, 16, 15, and 8 serotypes, respectively. Among four subtypes of EspP encoded by these alleles, EspPα (produced by enterohemorrhagic E. coli [EHEC] O157:H7 and the major non-O157 EHEC serotypes) and EspPγ cleaved pepsin A, human coagulation factor V, and an oligopeptide alanine-alanine-proline-leucine-para-nitroaniline, whereas EspPβ and EspPδ either were not secreted or were proteolytically inactive. The lack of proteolysis correlated with point mutations near the active serine protease site. We conclude that espP is widely distributed among STEC strains and displays genetic heterogeneity, which can be used for subtyping and which affects EspP activity. The presence of proteolytically active EspP in EHEC serogroups O157, O26, O111, and O145, which are bona fide human pathogens, suggests that EspP might play a role as an EHEC virulence factor.     

High affinity nanobodies against human epidermal growth factor receptor selected on cells by E. coli display

Most therapeutic antibodies (Abs) target cell surface proteins on tumor and immune cells. Cloning of Ab gene libraries in E. coli and their display on bacteriophages is commonly used to select novel therapeutic Abs binding target antigens, either purified or expressed on cells. However, the sticky nature of bacteriophages renders phage display selections on cells challenging. We previously reported an E. coli display system for expression of VHHs (i.e., nanobodies, Nbs) on the surface of bacteria and selection of high-affinity clones by magnetic cell sorting (MACS). Here, we demonstrate that E. coli display is also an attractive method for isolation of Nbs against cell surface antigens, such as the epidermal growth factor receptor (EGFR), upon direct selection and screening of Ab libraries on live cells. We employ a whole cell-based strategy using a VHH library obtained by immunization with human tumor cells over-expressing EGFR (i.e., A431), and selection of bacterial clones bound to murine fibroblast NIH-3T3 cells transfected with human EGFR, after depletion of non-specific clones on untransfected cells. This strategy resulted in the isolation of high-affinity Nbs binding distinct epitopes of EGFR, including Nbs competing with the ligand, EGF, as characterized by flow cytometry of bacteria displaying the Nbs and binding assays with purified Nbs using surface plasmon resonance. Hence, our study demonstrates that E. coli display of VHH libraries and selection on cells enables efficient isolation and characterization of high-affinity Nbs against cell surface antigens.     

The Serine Protease EspC from Enteropathogenic Escherichia coli Regulates Pore Formation and Cytotoxicity Mediated by the Type III Secretion System

Type III secretion systems (T3SSs) are specialized macromolecular machines critical for bacterial virulence, and allowing the injection of bacterial effectors into host cells. The T3SS-dependent injection process requires the prior insertion of a protein complex, the translocon, into host cell membranes consisting of two-T3SS hydrophobic proteins, associated with pore-forming activity. In all described T3SS to date, a hydrophilic protein connects one hydrophobic component to the T3SS needle, presumably insuring the continuum between the hollow needle and the translocon. In the case of Enteropathogenic Escherichia coli (EPEC), the hydrophilic component EspA polymerizes into a filament connecting the T3SS needle to the translocon composed of the EspB and EspD hydrophobic proteins. Here, we identify EspA and EspD as targets of EspC, a serine protease autotransporter of Enterobacteriaceae (SPATE). We found that in vitro, EspC preferentially targets EspA associated with EspD, but was less efficient at proteolyzing EspA alone. Consistently, we found that EspC did not regulate EspA filaments at the surface of primed bacteria that was devoid of EspD, but controlled the levels of EspD and EspA secreted in vitro or upon cell contact. While still proficient for T3SS-mediated injection of bacterial effectors and cytoskeletal reorganization, an espC mutant showed increased levels of cell-associated EspA and EspD, as well as increased pore formation activity associated with cytotoxicity. EspP from enterohaemorrhagic E. coli (EHEC) also targeted translocator components and its activity was interchangeable with that of EspC, suggesting a common and important function of these SPATEs. These findings reveal a novel regulatory mechanism of T3SS-mediated pore formation and cytotoxicity control during EPEC/EHEC infection.     

Molecular optimization of autotransporter-based tyrosinase surface display

Display of recombinant enzymes on the cell surface of Gram-negative bacteria is a desirable feature with applications in whole-cell biocatalysis, affinity screening and degradation of environmental pollutants. One common technique for recombinant protein display on the Escherichia coli surface is autotransport. Successful autotransport of an enzyme largely depends on the following: (1) the size, sequence and structure of the displayed protein, (2) the cultivation conditions, and (3) the choice of the autotransporter expression system. Common problems with autotransporter-mediated surface display include low expression levels and truncated fusion proteins, which both limit the cell-specific activity. The present study investigated an autotransporter expression system for improved display of tyrosinase on the surface of E. coli by evaluating different variants of the autotransporter vector including: promoter region, signal peptide, the recombinant passenger, linker regions, and the autotransporter translocation unit itself. The impact of these changes on translocation to the cell surface was monitored by the cell-specific activity as well as antibody-based flow cytometric analysis of full-length and degraded passenger. Applying these strategies, the amount of displayed full-length tyrosinase on the cell surface was increased, resulting in an overall 5-fold increase of activity as compared to the initial autotransport expression system. Surprisingly, heterologous expression using 7 different translocation units all resulted in functional expression and only differed 1.6-fold in activity. This study provides a basis for broadening of the range of proteins that can be surface displayed and the development of new autotransporter-based processes in industrial-scale whole-cell biocatalysis.     

Autodisplay of an archaeal γ-lactamase on the cell surface of Escherichia coli using Xcc_Est as an anchoring scaffold and its application for preparation of the enantiopure antiviral drug intermediate (−) vince lactam

At present, autotransporter protein mediated surface display has opened a new dimension in the development of whole-cell biocatalysts. Here, we report the identification of a novel autotransporter Xcc_Est from Xanthomonas campestris pv campestris 8004 by bioinformatic analysis and application of Xcc_Est as an anchoring motif for surface display of γ-lactamase (Gla) from thermophilic archaeon Sulfolobus solfataricus P2 in Escherichia coli. The localization of γ-lactamase in the cell envelope was monitored by Western blot, activity assay and flow cytometry analysis. Either the full-length or truncated Xcc_Est could efficiently transport γ-lactamase to the cell surface. Compared with the free enzyme, the displayed γ-lactamase exhibited optimum temperature of 30 °C other than 90 °C, with a substantial decrease of 60 °C. Under the preparation system, the engineered E. coli with autodisplayed γ-lactamase converted 100 g racemic vince lactam to produce 49.2 g (?) vince lactam at 30 °C within 4 h. By using chiral HPLC, the ee value of the produced (?) vince lactam was determined to be 99.5 %. The whole-cell biocatalyst exhibited excellent stability under the operational conditions. Our results indicate that the E. coli with surface displayed γ-lactamase is an efficient and economical whole cell biocatalyst for preparing the antiviral drug intermediate (?) vince lactam at mild temperature, eliminating expensive energy cost performed at high temperature.     

Grimelysin, a novel metalloprotease from Serratia grimesii, is similar to ECP32

Limited actin proteolysis is the hallmark of bacterial metalloprotease ECP32. While ECP32 has long been considered an Escherichia coli protein, the N-terminal amino acid sequence of the active enzyme described previously, could not been retrieved in the E. coli genome. We cloned, sequenced and characterized Serratia grimesii protease grimelysin and show that grimelysin is similar to the previously described protease ECP32.     

The role of the catalytic domain of E. coli GluRS in tRNA discrimination

Discrimination of tRNA^Gln is an integral function of several bacterial glutamyl-tRNA synthetases (GluRS). The origin of the discrimination is thought to arise from unfavorable interactions between tRNA^Gln and the anticodon-binding domain of GluRS. From experiments on an anticodon-binding domain truncated Escherichia coli (E. coli) GluRS (catalytic domain) and a chimeric protein, constructed from the catalytic domain of E. coli GluRS and the anticodon-binding domain of E. coli glutaminyl-tRNA synthetase (GlnRS), we show that both proteins discriminate against E. coli tRNA^Gln. Our results demonstrate that in addition to the anticodon-binding domain, tRNA^Gln discriminatory elements may be present in the catalytic domain in E. coli GluRS as well.     

Autodisplay of an avidin with biotin-binding activity on the surface of <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To display a recombinant avidin fused to the autotransporter ShdA to bind biotinylated molecules on the surface of Escherichia coli.

Results

Two chimeric protein constructs containing avidin fused to the autotransporter ShdA were expressed on the surface of Escherichia coli DH5α. One fusion protein contained 476 amino acids of the ShdA α and β domains, whereas the second consisted of a 314 amino acid from α and truncated β domains. Protein production was verified by SDS-PAGE using an antibody to the molecular FLAG-tag. The surface display of the avidin-shdA fusion protein was confirmed by confocal microscopy and flow cytometry analysis, and the biotin-binding activity was evaluated by fluorescence microscopy and flow cytometry using biotin-4-fluorescein and biotinylated-ovalbumin (OVA).

Conclusions

Expression of a recombinant avidin with biotin-binding activity on the surface of E. coli was achieved using the autotransporter ShdA. This system is an alternative to bind biotinylated molecules to E. coli.