key: cord-0910061-wazfxp50
authors: Gautam, Samir; Spiegel, David A.
title: Pushing the Bacterial Envelope: Strategies for Re-Engineering Bacterial Surfaces with Heterologous Proteins and Sugars
date: 2014-06-06
journal: Micro- and Nanoengineering of the Cell Surface
DOI: 10.1016/b978-1-4557-3146-6.00004-0
sha: f4db6aef4664468b856f4029f5c8f6be6c887e61
doc_id: 910061
cord_uid: wazfxp50

Over the past three decades, a powerful array of techniques has been developed for expressing heterologous proteins and saccharides on the surface of bacteria. Surface-engineered bacteria, in turn, have proven useful in a variety of settings, including high-throughput screening, biofuel production, and vaccinology. In this chapter, we provide a comprehensive review of methods for displaying polypeptides and sugars on the bacterial cell surface, and discuss the many innovative applications these methods have found to date. While already an important biotechnological tool, we believe bacterial surface display may be further improved through integration with emerging methodology in other fields, such as protein engineering and synthetic chemistry. Ultimately, we envision bacterial display becoming a multidisciplinary platform with the potential to transform basic and applied research in bacteriology, biotechnology, and biomedicine.

The bacterial envelope is a multilayered organelle that surrounds the cell, providing morphological structure and protection from environmental stressors. In gram-negative organisms, the envelope comprises an inner membrane (IM), an outer membrane (OM), and a periplasmic space in between that contains the peptidoglycan cell wall-a highly cross-linked mesh-like macromolecule that confers mechanical strength to the cell (Figure 4 .1A). Grampositive bacteria, in contrast, lack an OM and have a thicker, surface-exposed cell wall (Figure 4 .1B). These structures have been studied intensely for over 60 years by bacterial cell biologists and antibiotic developers, among others, due to their critical roles in bacterial growth and survival [1, 2] .

As our understanding of the bacterial envelope has grown, however, so has its utility; we now possess a powerful set of techniques for expressing proteins on the cell surface that has transformed the envelope from a target of basic and pharmaceutical research into a versatile platform for biotechnological innovation. Since the first examples in 1986 [3, 4] , protein display in bacteria has matured into a robust, streamlined methodology with an expansive range of applications, including environmental remediation [5] , biofuel production [6] , biocatalysis [6, 7] , biosensing [8] , protein library screening [9] , cancer therapy [10] , and vaccinology [7,11À14] .

More recently, bacterial surface engineering has extended beyond the polypeptide paradigm to include sugars as well. This technology, referred to as "glycoengineering," [15À17] involves heterologous expression of genes that mediate saccharide biosynthesis, enabling the display of a variety of nonnative proteoglycans, glycolipids, and polysaccharides on the surface of recombinant bacteria. Glycoengineered organisms, in turn, have been utilized as whole cell vaccines [18À21], anti-infective probiotics [22] , and "living factories" for glycoconjugate synthesis [16] .

In this chapter, we describe the repertoire of techniques that have been established for displaying heterologous proteins and sugars on the surface of bacteria. We then discuss how re-engineered bacteria have been exploited in basic and applied biology thus far. Throughout, we attempt to highlight recently developed technologies across disciplines that may prove useful in the context of bacterial surface display, improving existing applications and perhaps enabling new ones. Finally, we address an important caveat: despite the tantalizing clinical and biotechnological prospects for surface-engineered bacteria, their implementation outside the laboratory has been greatly hindered due to concerns over the environmental dangers of genetically modified organisms (GMOs). We therefore include a discussion of these theoretical risks and present a set of recombinant techniques for circumventing them (see Box 4.1) . 

A number of potentially transformative applications have been devised for surfaceengineered bacteria, but their practical use has been relatively limited due to four major concerns about the potential hazards of releasing GMOs into the environment [23À25] .

First is the risk of parallel transfer of foreign DNA to other organisms, a particular issue in the case of virulence gene expression in vaccine vectors. This risk can be minimized by increasing the stability of the genetic modification, both through chromosomal integration of the foreign DNA, and by ensuring that the chromosomal insertion site is distant from especially mobile genetic elements such as transposons.

Second is the possibility of releasing an organism with significant fitness advantage, leading to unrestrained propagation of the GMO and damaging environmental consequences. Fears of this outcome may be allayed through the use of auxotrophic strains unable to survive in the environment (i.e., "biologically contained" organisms) or through use of non-living vehicles such as OMVs, BGs, and inactivated spores.

Third is a risk unique to vaccine delivery: the possibility of an attenuated vaccine strain reverting to a virulent genotype, thus placing the vaccinee and/or close contacts in danger of contracting disease. Attenuation is usually accomplished by disrupting a gene involved with MICRO-AND NANOENGINEERING OF THE CELL SURFACE

The majority of bacterial protein display methods employ a common strategy in which a heterologous protein of interest (passenger) is genetically fused to an endogenous surface protein (carrier). The structure of the cell envelope, in turn, determines where these extracellular passengerÀcarrier chimeras reside; in gram-negative organisms they associate with the OM (Figure 4 .1A), while in gram-positive bacteria chimeras are usually attached to the peptidoglycan cell wall (Figure 4 .1B) [30] . either virulence or central metabolic pathways. The former is associated with greater risk as virulence factors are generally located on mobilizable genetic elements that may be acquired from wildtype organisms. Metabolic genes, meanwhile, are generally located on the chromosome, so attenuations at these sites are more stable [23] .

The fourth concern-introduction of drug resistance genes into the environment-is perhaps the most worrisome. In the laboratory setting, maintaining heterologous genes episomally in bacteria using antibiotic resistance selection markers (the simplest of recombinant techniques) is perfectly acceptable practice. However, it is imperative for genetically engineered bacteria released into the environment-such as vaccines and bioremediation devices-to be free of such markers, so as to prevent spread of drug resistance. Two genetic methods that satisfy this requirement have been established: the balanced lethal system (more widely used) [26, 27] and recombineering [28] -both of which are effective in gram-negative and gram-positive [29] organisms.

The balanced lethal approach uses an episomal plasmid to express the gene of interest, but importantly, the plasmid is maintained through complementation rather than antibiotic resistance [26, 27] . Generation of a markerless carrier strain using the balanced lethal system proceeds as follows. A key metabolic gene such as one involved in amino acid synthesis is deleted, rendering the strain auxotrophic. This genetic deletion is achieved through (i) heterologous recombination between the host chromosome and a suicide vector encoding a null allele and a resistance marker flanked by recombinase target sites, (ii) antibiotic-mediated selection for transformants, and (iii) subsequent excision of the resistance marker through transient expression of recombinase. (Note: markerless strains may be identified using a negative selection marker such as sacB, which confers sensitivity to sucrose.) The mutant strain is then transformed with a plasmid containing two elements: the gene of interest and the metabolic gene deleted from the host. Thus, the episomal element is stably maintained in the recombinant strain, as its loss would remove complementation with the essential gene.

In recombineering, stabilization of the gene of interest is guaranteed by direct integration into the bacterial chromosome [28] . This is achieved through the use of a modified strain expressing the lamba-red bacteriophage system-a set of enzymes that greatly increase the efficiency of homologous recombination. After induction of the recombination enzymes, the strain is electroporated with a linear DNA construct that contains (i) the heterologous gene, (ii) flanks consisting of sequences complementary to the chromosomal insertion site, and (iii) a selection marker which is itself flanked by recombinase target sites. After selection for integrants, the marker is removed using recombinase to produce the desired strain.

A number of recombinant techniques have been developed for expressing passengerÀcarrier fusion proteins on the surface of gramnegative species (Figure 4 .1A). For peptides and small proteins up to approximately 60 amino acids [31] , exogenous sequences may be inserted into (i) proteins that comprise surface appendages such as pili and flagella, (ii) extracellular loops of outer membrane proteins such as LamB and OmpA, or (iii) lipoproteins and virulence factors anchored to the surface [13, 32] . However, for the display of larger proteins such as enzymes, passengers must be fused to the N-or C-terminus of carriers rather than intracistronically (Figure 4 .1C,iii) [13] .

Autodisplay is one such system commonly used for the display of large proteins. The strategy involves fusion of passenger proteins to autotransporters, a class of virulence factors that includes IgA1 protease from Neisseria gonorrhoeae, MisL from Salmonella enterica serovar Typhimurium, and AIDA-I (adhesin involved in diffuse adherence) from Escherichia coli [7] . During normal expression, autotransporters insert into the OM, forming a porinlike β-barrel structure with their C-terminal domain. The N-terminal domain, connected to the β-barrel via a long, flexible linker is subsequently translocated through the pore to access the extracellular milieu. Therefore, autotransporters may be 'hijacked' to allow surface display by replacing the native N-terminal domain with a heterologous passenger [33] . A notable advantage of this approach is the lateral mobility of carriers within the OM, which enables multimerization of passenger proteins, as has been demonstrated with surface-displayed streptavidin [34] , nitrilase from Klebsiella pneumoniae [35] , and sorbitol dehydrogenase [36] . The other major system for expressing large proteins at the bacterial surface involves fusion to ice nucleation protein (INP), an abundant lipoprotein present in the OM of many plant pathogens [13, 37] . A unique property of INP is its mechanism of surface expression; rather than threading through the IM in an unfolded state like the majority of bacterial proteins, INP is translocated fully folded. Thus, INPmediated surface display can accommodate passenger proteins that associate with bulky cofactors, such as heme-and diflavincontaining cytochrome P450 enzymes [13, 38] .

Since their introduction in the 1990s [39, 40] , autodisplay and INP-based expression systems have developed into highly optimized, well-characterized biotechnological platforms. PassengerÀcarrier chimeras expressed using these methods (i) efficiently traverse the IM and periplasm to reach the cell surface in great abundance (more than 100,000 molecules per cell in the case of autodisplay [41] ), (ii) reside stably at the surface without substantial detachment and loss of the recombinant protein, (iii) resist degradation by proteases within the periplasmic space, and (iv) do not disrupt the integrity of the cell envelope or induce growth defects [42] . Given these properties, along with the well-defined genetic techniques required for autodisplay-and INP-based expression, surface display in gram-negative bacteria can be considered a straightforward and robust class of techniques.

Whereas surface display in gram-negative bacteria is achieved through fusion to carrier proteins expressed on the OM, a different strategy must be undertaken in gram-positive organisms, which lack this outer vestment ( Figure 4 .1B). The most common method involves attaching proteins directly to the surface-exposed peptidoglycan cell wall, using a class of enzymes called sortases (Figure 4 .1B,i) [43, 44] . These membraneassociated enzymes recognize specific pentapeptide sorting motifs in secreted proteins, cleave within the motif, and covalently attach the N-terminal portion to the cell wall [45] . Thus, fusion of a passenger protein to the N-terminus of a sortase substrate protein enables efficient expression on the grampositive cell surface. Sortase-mediated display of heterologous proteins has proven especially robust both because of the covalent nature of attachment, and because of the rigid peptidoglycan substrate, which ensures stability of surface expression [11] .

Alternatives to sortase-based surface display have been explored as well. For example, passengers fused to transmembrane proteins that extend from the plasma membrane through the cell wall and into the extracellular space are effectively displayed at the cell surface ( Figure 4 .1B,ii) [46] . Surface expression can also be achieved using chimeras between passengers and proteins that comprise the bacterial cell surface layer (S-layer), a self-assembling crystalline lattice that coats several species of bacteria ( Figure 4 .1B,iii) [47À52] . Notably, the absence of an OM on gram-positive organisms removes a significant obstacle to protein translocation, facilitating the display of large proteins when using sortase-mediated, transmembrane fusion, and S-layer fusion protein methodologies [11] .

A final, somewhat unique approach involves fusion to autolysins-cell wall degradative enzymes that bind non-covalently to components of the gram-positive cell wall (Figure 4 .1B,iv) [53À57]. The key advantage of this methodology is that autolysin-passenger chimeras may be expressed in a model organism (e.g., E. coli), purified, and adsorbed to the cell wall of a wild-type gram-positive strain. Thus, using this strategy, gram-positive bacteria can be re-engineered with heterologous surface proteins, but remain genetically unchanged, thereby circumventing the potential environmental risks of GMOs (see Box 4.1) [55] .

Certain bacterial species, such as those within the Clostridium and Bacillus genera, display a unique behavior known as sporulation, in which bacteria transform from metabolically active (vegetative) cells into dormant structures (spores) in response to nutrient depleted conditions [12] . During this process, bacterial DNA becomes cocooned within a multilayered wall of peptidoglycan and concentric protein shells, rendering the DNA impervious to toxic chemicals, radiation, and extremes of pH, temperature, and dehydration [12] . Spores are thus able to survive in inhospitable environments for extended periods-in one report, 25 million years [12, 58] . Despite the spore's extraordinary inertness, its surface proteins remain functional (in part due to their attachment to a stable matrix, see Section 4.3.4), ready to sense nutrient repletion, and germinate into a fully active state [59] . Numerous methods have been established for displaying heterologous proteins on these remarkable structures, all involving fusion to carrier proteins expressed at the spore surface [12] . These carriers include CotB, CotC, and CotG in Bacillus subtilis; [60] . BclA and BclB in Bacillus anthracis [61] ; and Cry1Ac in Bacillus thuringiensis [62] . Spores re-engineered by these means have proven useful as biosensors [8,63À69] , biocatalysts [70] , and vaccines [71, 72] .

While the applications of spores are discussed in more detail below, it is worth describing here the unique advantages spores offer over conventional bacteria in three biotechnological settings. First, similar to vegetative cell systems, spores are well suited to biosensing-the detection and reporting of analytes in solution (see Section 4.3.5.1)because of their sensitivity, low cost, ease of use, and small size. The exceptional stability of spores, however, allows them to be used outside of the laboratory in harsh natural environments, where conventional cellular systems cannot survive [70] . Second, spores are unique in that proteins need not traverse a membrane in order to be expressed on the cell surface; instead, proteins are attached to the outermost layer of the spore, which is assembled within the cytoplasm of the mother cell during sporulation [73] . This property allows expression of multimers such as streptavidin [74] , proteins with problematic hydrophobic domains that preclude membrane translocation [60] , enzymes requiring cofactors [60] , and very large proteins [31] . Finally, the spore's ability to germinate confers an advantage in the context of vaccine delivery, as it remains stable during storage at room temperature, but then transforms once delivered to the nutrientrich environments of the tissue into vegetative cells with potent immunostimulatory properties (see Section 4.3.1) [75, 76] .

Outer membrane vesicles (OMVs) and bacterial ghosts (BGs) are acellular derivatives of the gram-negative envelope that contain the parent cell's surface-expressed proteins [77À79]. OMVs consist of outer membrane-derived bilamellar vesicles, 50À250 nm in diameter, which are continuously elaborated by growing gram-negative bacteria [77] . Efficient surface display on OMVs has been achieved by fusing passenger proteins to the E. coli toxin ClyA, which becomes concentrated on these structures during vesiculation [80] . OMVs have particular utility as vaccine delivery vehicles because they are stable during prolonged storage [81] , and strongly immunogenic (by virtue of the many immunostimulatory components of the OM these vesicles retain), but do not present the same environmental safety hazards as intact cells because they are unable to propagate (see fusion of the two membranes. Protein E oligomerization leads to the formation of a "lysis tunnel" (Figure 4 .2C), through which the cytoplasm is released extracellularly. However, due to the controlled nature of the lytic event and the effective seal between IM and OM, cell envelope morphology and periplasmic content are maintained [78] . Most commonly, anchoring of foreign proteins on BGs is achieved through fusion of passengers with the outer membrane protein, OmpA. Finally, like OMVs, BGs are excellent vehicles for vaccine delivery because of their potent immunostimulatory properties and inability to replicate [85, 86] .

Bacterial surface display has lent itself to a host of biotechnological and biomedical applications. These can be organized into four categories: (i) display of antigens in vaccine design, (ii) expression of antitumor proteins for cancer therapy, (iii) immobilization of enzymes for biocatalysis, and (iv) a set of related applications that involve binding between surface proteins and molecules in solution (biosensing, biosorption, and library screening). These topics will be discussed here only in brief; please see focused reviews for greater detail [6,9,11À13 ].

In general, vaccines have two components: a pathogen-associated antigen that serves as the target for adaptive immune responses and an adjuvant-a material that potentiates and tunes immune responses to the co-delivered antigen [87] . Uptake of these components by antigen presenting cells (APCs) such as dendritic cells represents the crucial initiating step in the generation of immune responses that protect against future infection.

Surface-engineered bacterial vaccines are constructed by expressing a pathogenassociated antigen on an inactivated bacterium, live-attenuated pathogen, or food grade (nonpathogenic) bacterial species. The bacterial vector itself is able to serve as an adjuvant due to the numerous immunostimulatory pathogen-associated molecular patterns (PAMPs) present within the cytoplasm and cell envelope. These include bacterial DNA, peptidoglycan, gram-positive lipoproteins, gram-negative lipopolysaccharide (LPS), and a set of PAMPs associated with viable cells (vita-PAMPs) which includes mRNA and cyclic dinucleotides [88] .

Localization of antigen at the bacterial surface per se appears to be important for the immunogenicity of these recombinant vaccine vectors. In almost all direct comparisons between surface, cytoplasmic, and secreted expression systems, surface-engineered vaccines have proven most efficacious (even when normalized to magnitude of expression) [89À100]. One reason for the improved immunogenicity of surface-anchored antigen is that it is efficiently co-delivered with adjuvant (bacterial PAMPs) to APCs, which is essential for generating effective immune responses [101] . Furthermore, surface expression produces a multivalent display of antigen on the cell surface, which is known to promote crosslinking of B-cell receptors and stimulation of humoral immune responses [102] .

Bacterial surface display has been especially useful in the development of mucosal vaccines with several advancing as far as clinical trials [7,11À14] . Table 4 .1 provides a summary of the bacterial vectors used in mucosal vaccinology, and a catalog of the pathogens these vaccines have targeted.

Several arguments have been proposed for why surface-engineered bacteria have proven such effective mucosal immunogens. The first derives simply from the size of the bacterial vector, which at .500 nm in diameter is considered "particulate." Delivery of antigen by such particulate vehicles is known to greatly improve antigen presentation by APCs, leading to stronger elicitation of immune responses at the mucosa than the same antigen in soluble form [88] . Second, the intrinsic adjuvanticity of bacteria provides a potent immunostimulatory signal to overcome the bias toward tolerance that exists in mucosal immune tissue [212] .

Third, the transient colonization and/or mild infection induced by live bacterial vaccines leads to a sustained, local generation of antigen at the mucosa that greatly improves delivery to APCs [213] . Finally, bacterial vaccines are uniquely efficient at gaining access to APCs within mucosal immune tissue, by virtue of surface properties that prevent trapping within the mucus layer [214] , and surface adhesion proteins and carbohydrates that promote attachment to mucosal epithelium and translocation into immune inductive sites [215, 216] . With respect to bacterial surface display, it is worth noting that surface expression of targeting proteins, such as antibodies that bind to proteins on epithelial cells, has been shown to further increase adhesion and translocation of bacterial vaccine vectors into immune tissue [193, 217] .

The use of live bacteria in cancer therapy has fascinated researchers since the early nineteenth century, when tumor regression was observed in a set of cancer patients after they contracted gas gangrene (an aggressive soft tissue infection caused by C. perfringens) [10,218À220] . The anticancer effect of bacteria has since been traced to a number of intrinsic properties of these organisms, such as their ability to home to tumor tissue with up to 1000-fold selectivity over healthy tissues [221] . In the case of C. perfringens, this ability arises from the fact that spores selectively germinate within the core of solid tumors, which is hypoxic due to poor vascularization [222] . Facultative anaerobes like Salmonella species and L. monocytogenes, meanwhile, accumulate in cancer tissue due to entrapment in the tortuous tumor vasculature [223] , chemotaxis toward nutrients released from dying cancer cells [224] , and preferential growth in the tumor's unique metabolic milieu [224] , where they are also protected from attack by white blood cells due to local immunosuppression [225, 226] . Upon accumulation in the tumor, bacteria promote cell death both by competing for nutrients and stimulating normally suppressed intratumoral immune cells to attack cancer cells [225] .

With the advent of genomics and recombinant technology, antitumor bacteria-which had largely remained curiosities in the field of cancer treatment-have now been transformed into finely tuned therapeutic devices through surface expression of proteins with antitumoral properties. For instance, the invasin adhesion protein from Y. pseudotuberculosis, which mediates entry of the bacterium into nonphagocytic mammalian cells, has been expressed on the surface of nonpathogenic E. coli-based therapeutic strains to promote invasion into cancer cells [227] . This strategy was utilized to deliver "trans-kingdom" RNAi in a mouse model of colon cancer to silence expression of the epithelial oncoprotein, β-catenin [228] . In another example, S. enterica serovar Typhimurium was engineered to express the apoptosis-promoting protein, Fas ligand, on its cell surface. The modified bacterium was shown to inhibit growth of primary breast and colon cancers as well as pulmonary metastases in mice [229] . Finally, bacteria have been engineered to express cytotoxic prokaryotic proteins such as cytolysin A, a surface-localized pore-forming toxin, producing strains that significantly inhibit tumor growth and metastasis in murine models of colon and lung cancer [230, 231] .

A number of other non-surface related genetic modifications have been shown to potentiate the anticancer effects of bacterial vectors as well, many of which could conceivably be combined with surface display to create more effective, multimodal therapeutics. For instance, Salmonella spp. have been designed to secrete pro-inflammatory cytokines such as IL-2 that promote adaptive immune responses [232] , as well as cytotoxic agents such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which directly induce tumor lysis [233] . A second effective modification is expression of genetic circuits that selectively activate gene expression in response to cues in the tumor microenvironment [234] . Such circuits have been used to couple cytotoxin production to arrival at tumors, which reduces the off-target toxicity of these tissue-damaging proteins. Third, through expression of prodrug converting enzymes, tumor-localized bacteria can be used to activate chemotherapeutic prodrugs in situ, increasing drug concentrations in the poorly accessible tumor core, and reducing collateral damage to healthy tissue. The efficacy of this approach has been confirmed not only in murine models but also in patients as well, as phase I trials have shown that an attenuated Salmonella strain, VNP20009, expressing the E. coli cytosine deaminase genewhich converts the prodrug 5-fluorocytosine into the active chemotherapeutic 5-fluorouracil (5-FU)-homed to tumors and generated significantly elevated intratumoral 5-FU levels with respect to serum [235] .

Of course, intravenous administration of live bacteria is not without risk of side effects. For instance, preliminary tolerability studies with VNP20009 showed dose-dependent elicitation of cytokines, such as IL-1β, IL-6, and TNF-α, which were associated with hypotension, fever, thrombocytopenia, anemia, diarrhea, nausea, and vomiting [236] . However, through detoxification of pro-inflammatory bacterial components such as LPS (see Section 4.3.1), attenuating mutations to virulence factors and genes in central metabolic pathways (see Box 4.1), titration of tolerable doses, and careful clinical monitoring postadministration, the immunotoxicity of live bacteria may be minimized, allowing these potentially valuable therapeutics to be tested further in clinical settings.

In addition to serving as devices designed to directly kill cancer cells, genetically engineered bacteria have also proven a highly effective platform for development of anticancer vaccines [237À241]. In actuality, these two approaches overlap in many ways; for example, vaccine vectors such as L. monocytogenes colonize tumors and induce direct cytotoxicity, while conversely, oncotherapeutic bacteria lead not only to tumor damage but also to tumor-specific adaptive responses due to release of cancer cell antigens during bacterially induced lysis [240, 242] . Nevertheless, in cancer vaccinology, elicitation of immune responses is the primary objective, the ultimate goal being immune-mediated clearance of both primary tumors and metastases as well. In this context, the immunostimulatory properties of the live bacterial vector are leveraged to break the inherent tolerance to cancer-associated antigens that exists because of their similarity to self proteins and their residence in an immunosuppressive tumor milieu [243] . Notably, translation of bacteria-based anticancer vaccines to the clinic has recently begun, with phase I trials demonstrating the safety of a promising listerial vaccine against cervical carcinoma [244] .

Bacterial surface display has been used in the design of anticancer vaccines extensively as a means of antigen expression. For instance, T-cell epitopes from NY-ESO-1, a commonly expressed tumor antigen, were inserted into the fimbrial proteins of S. enterica serovar Typhimurium to create a vaccine capable of generating robust antigen-specific T-cell responses in mice [245] . Similarly, the E7 oncoprotein (which is involved with the development of cervical cancer) and the 37 kDa oncofetoprotein (which is found in many human cancers) were surface expressed on lactic acid bacteria to generate effective mucosal vaccines against human papillomavirusinduced tumors [246À249]. Further, it was shown in a mouse model of cervical cancer that the immunogenicity of live E7-expressing bacterial vaccines was greatly potentiated when the vector simultaneously secreted the pro-inflammatory cytokine, IL-12 [155] . This important finding illustrates the potential of combining multiple recombinant modifications in the same therapeutic. It also suggests that vaccine-generated cytokine secretion is a strategy that could, in principle, boost the immunogenicity of any live vaccine.

In addition to antigen expression, bacterial surface display has been used to improve targeting of bacterial vectors to tumors, in an analogous manner to the targeting of vaccines to mucosal immune tissue (described in Section 4.3.1). On an attenuated Salmonella vaccine strain, for example, surface display of single chain antibody fragments specific for carcinoembryonic antigen-a glycoprotein overexpressed on many epithelial-derived cancers-was found to significantly improve intratumoral accumulation of the recombinant bacteria in vivo [250] .

The use of microbially expressed enzymes for (bio)catalysis of chemical reactions has a long history that includes the ancient practices of bread and cheese making, beer and wine fermentation, and, more recently, antibiotic production. The value of enzymes in chemical synthesis lies in their potential to catalyze reactions with remarkable rapidity in a highly chemo-, regio-, and enantioselective manner under mild conditions, while minimizing the toxic waste generation, protection/ deprotection steps, and energy expenditure involved in conventional synthetic chemistry [251, 252] . Furthermore, through directed evolution and other protein engineering techniques (described in Section 4.3.5.3), modified enzymes may be designed to (i) remain stable at elevated temperatures in solution with organic solvents, (ii) accept nonnative substrates, and (iii) catalyze novel reactions [251] . These properties have led to a rapidly growing interest in the use of biocatalysts in industrial production of detergents, food, cosmetics, textiles, fine chemicals, biofuels, and pharmaceuticals [253À255]. In fact, over 500 commercial products are produced using enzymes, generating an industrial enzyme market valued at US$5.1 billion as of 2009 [255] .

Immobilization of enzymes on the bacterial cell surface offers key advantages over the use of free enzyme in biocatalysis. First, attachment of enzymes to a solid matrix, such as the bacterial envelope, vastly increases their stability [7, 256] ; in one example, a surface-expressed organophosphorus hydrolase retained 100% catalytic efficiency for an astounding 45 days in culture [257] . Second, the production of enzyme on the cell surface obviates the need for protein purification, which can be laborious and costly. Third, once genetic engineering of a biocatalyst strain is complete, there is access to a nearly infinite supply of enzyme, requiring only culture in inexpensive growth medium-again greatly reducing the price of production. Enzyme display on the cell surface also has advantages over cytoplasmic expression, in that it allows use of substrates and generation of products that are impermeable to lipid membranes.

A wide array of enzymes have been expressed on the surface of bacteria, including oxidoreductases (e.g., cytochrome P450 enzymes [38, 258] ) and various hydrolases (e.g., glycosyltransferases [259] , phosphatases [260] , lipases [261] , other esterases [262] ) [13, 33] . Organisms re-engineered with such enzymes have been used to degrade organic pollutants, generate fuels from renewable energy sources (e.g., plant biomass), and manufacture chemical compounds for pharmaceutical and industrial purposes [6] .

In this section, we discuss applications of bacterial surface display that are based on binding between a surface-bound protein and a molecule in solution, which may be a protein, a small molecule, or an element. A myriad of inventive uses for this binding interaction have been established, falling under the headings of biosensing, biosorption, and screening (Figure 4 .3).

Over the last two decades, researchers have endeavored to genetically engineered bacteria that serve as devices for detecting and quantifying molecules in solution. To this end, an entire class of "two-component biosensors" (Figure 4 .3A) has been devised. These bacteria express (i) a genetic sensor that binds to an analyte and transduces the binding event into a dose-dependent change in activity of a promoter and (ii) a reporter gene driven by said promoter that produces a measurable signal (e.g., luciferase, green fluorescent protein, or β-galactosidase) [263] . In one notable example, E. coli was converted into a biosensor for arsenite-containing compounds through transformation with a plasmid containing the ars operon, which mediates bacterial resistance to such toxins. This regulatory circuit works as follows: in the absence of arsenite, the protein arsR represses the ars promoter, but upon binding the metalloid, it unbinds the promoter to permit expression of several genes that detoxify and extrude arsenite-containing compounds. Thus, by expressing a promoterless bacterial luciferase (luxAB) gene [264] downstream of the ars promoter in E. coli, a simple biological sensor for arsenite could be constructed. Remarkably, due to the sensitivity of arsR for its substrate and the signal amplification inherent in such operons, this re-engineered bacterial strain was capable of detecting arsenite at subattomolar concentrations-1000 times below the detection limits of conventional techniques [263, 265] . Similarly designed biosensors have been used to measure concentrations of (i) various heavy metals and organic toxins in the setting of environmental monitoring [266] , (ii) nutrients in the study of soil microbe metabolism [267] , and (iii) analytes with potential clinical relevance such as quorum-sensing molecules [268] and antimicrobial drugs [12, 269] .

To date, two-component biosensors have been constructed only with intracellular sensing mechanisms. However, surface-engineered bacteria have been used as the basis for a novel ELISA-like quantitative immunoassay. In this approach, single chain variable fragments (scFvs) of antibody proteins are expressed on the surface of bacteria, analogous to the immobilization of antibodies on ELISA plates. Samples are incubated with these recombinant bacteria, and antibody-bound analyte is then separated from unbound analyte via centrifugation. Quantification of bound analyte is performed using horseradish peroxidaseÀ antibody conjugates or other detection mechanisms. Along these lines, Chen et al. [270] have constructed scFv-expressing bacteria capable of measuring concentrations of the cardiac glycoside drug, digoxin, in the nanomolar range. The success of this approach indicates the enticing possibility that such diagnostic devices may complement or even replace traditional immunoassays like ELISA due to their cost efficiency, ability to self-regenerate, and relatively high sensitivity.

Bacteria expressing peptide or protein ligands for soluble binding partners have been used as effective chromatographic media for purification of a range of molecules (Figure 4 .3B). For example, biosorption of heavy metal toxins to metal-binding peptides on the surface of E. coli and Staphylococcus species has been used for environmental remediation of polluted water [5] . Meanwhile, expression of peptide epitopes has enabled isolation of single antibody clones from polyclonal serum samples (produced in immunized rabbits)-an approach that may serve as an alternative to hybridoma development for accessing monoclonal antibodies [271] .

The scope of this technology, in fact, is likely to broaden further in light of a recent landmark study in protein engineering, which showed that novel proteins capable of binding to ligands of interest can be rationally designed in silico [272] . The authors of this study described a general computational method for designing small molecule-binding sites and applied it to create a protein capable of binding to the steroid digoxigenin. Through standard directed evolution methodology (described in Section 4.3.5.3), the group was able to obtain an optimized binder with picomolar affinity, and exquisite selectivity for digoxigenin over the related steroids digitoxigenin, progesterone, and β-estradiol. With this ability to program ligand specificity into synthetic proteins, it will, in theory, be possible to design biosensors and biosorbents for virtually any small molecule-a truly revolutionary advance.

Similar to the more commonly used phage display technique [273] , bacterial surface display enables the identification of proteins and peptides with desirable binding attributes by linking the identity of displayed proteins with their genetic sequence. To achieve this, large sets of heterogeneous bacterial clones are generated (by transforming bacteria with surface display vector libraries containing either cDNA or randomly mutated sequences), subjected to iterative rounds of screening and amplification, and sequenced to identify the surface protein(s) responsible for mediating binding interactions (Figure 4 .3C). The use of bacteria rather than phage in such screens is advantageous because of the density of surface protein expression, which, at .100,000 per cell, dwarfs the few molecules displayed per phage [274] , and greatly enhances the sensitivity of screens. Additional advantages are that bacteria, in contrast to phages, replicate autonomously and are of sufficient size to be detected by fluorescence-activated cell sorting (FACS) instruments, which allow quantitation of fluorescence of individual cells at rates upwards of 30,000 per second. This technology enables highly efficient identification of peptides and proteins with desired properties. For instance, through surface display of peptide libraries, the linear epitopes of antibodies have been rapidly determined through incubation of fluorescently labeled antibody with individual clones and FACS-mediated selection of antibody-bound bacteria (Figure 4 .3C) [275, 276] . Similar approaches have been taken to discover peptides with a range of targets including tumor cells [277, 278] , human signaling proteins [279, 280] , neural stem cells [281] , viral proteins [282] , etc. [9,283À287] . In addition, novel inhibitors for enzymes such as cathepsin G and trypsin have been culled from surface-displayed peptide libraries based on inhibitor enzyme binding affinities [288, 289] .

One particularly powerful application of combinatorial bacterial display is directed evolution, a process through which proteins are subjected to rounds of random mutagenesis and screening (often through FACS sorting) to identify "evolved" proteins with improved or unique function [290] . The use of bacteria is essential for directed evolution experiments because of their short generation time, amenability to genetic manipulation, and compatibility with sorting instruments.

Surface display-based directed evolution has found several applications thus far. In one example, antibodies with improved affinity were generated through anchored periplasmic expression (APEx) of antibody libraries in E. coli, permeabilization of the OM, incubation with fluorescent antigen, and FACS sorting of labeled cells [291À294]. Enzymes with novel activity have also been evolved using bacterial surface display [295À301]. Importantly, identification of hits in such screens differs from previously described methods, in that clones are not identified through interaction with fluorescent binding partners, but through attachment of fluorescent reaction products to the cell surface. For instance, in the development of an endopeptidase with novel substrate specificity, a library of bacterial clones displaying enzyme variants was incubated with a peptide substrate containing a BODIPY fluorophore on one side of the scissile bond and a quencher on the other. Clones expressing an enzyme capable of cleaving the bond would electrostatically capture an unquenched BODIPY-containing cleavage product on their membrane (by virtue of the product's 13 overall charge), allowing for rapid detection and isolation by FACS [298] .

As described above, surface display of exogenous proteins is accomplished through fusion of passengers to extracellular carriers. Heterologous expression of glycans, meanwhile, is more challenging because their biosynthesis is not template directed. Thus, installation of nonnative sugars on the bacterial cell surface must be achieved indirectly, via recombinant expression of enzymes that mediate synthesis, attachment, and remodeling of oligosaccharides, or deletion of endogenous genes involved with these processes (see Box 4.2 for a detailed description of endogenous bacterial glycans).

Three basic strategies have been used in bacterial glycoengineering. In the first, the operon encoding the entire metabolic pathway responsible for synthesis of a given sugar is cloned and transferred to a carrier strain (Figure 4.4A) . This approach has been used to express Pseudomonas aeruginosa capsular polysaccharide [18,307À309] and Shigella LPS [310À313] in a number of heterologous species. The second strategy involves site-specific N-linked glycosylation of target proteins (Figure 4.4A) . This is accomplished through expression of (i) the genes required for biosynthesis of the nonnative sugar; (ii) PglB, an oligosaccharyltransferase cloned from Campylobacter jejuni that catalyzes 

The surface glycome in gram-positive organisms consists of glycoproteins (such as S-layer proteins) [302] , secondary cell wall polysaccharides (which link the S-layer to peptidoglycan), teichoic acids (anionic glycopolymers which thread through the cell wall), and capsular polysaccharides (in those species expressing capsule) [303] . The biosynthesis of capsule, which represents one of the major targets of glycoengineering, takes place in the cytoplasm, where nucleotide phosphosugars are assembled into massive polymers of species-specific oligosaccharide repeat units by membraneassociated enzymes and translocated to the cell surface through dedicated transport pathways [304, 305] . These capsular polysaccharides shield bacteria from the immune system by preventing opsonization with antibodies and complement, consequently reducing phagocytosis by host immune cells.

Gram-negative bacteria also possess glycoproteins (such as pili [15] ) as well as capsular polysaccharides (in many species), but are unique in their expression of the complex glycolipid, LPS-another favorite target of glycoengineers (Figure 4.3) . LPS, also referred to as endotoxin for its ability to induce toxic shock in mammals, constitutes the OM of most gramnegative bacteria, and has three basic structural components (Figure 4 .3) [306] . Lipid A, which is highly conserved across genera and mediates endotoxicity, represents the hydrophobic moiety that anchors LPS to the membrane. The attachment of glycans to an asparagine residue within the glycosylation sequon, D/EYNXS/T; and (iii) a modified surface protein containing the heptapeptide glycosylation sequon [15À17] . A third strategy entails glycan remodeling through addition and/or deletion of enzymes that modify cell surface oligosaccharides-most commonly LPS, as described in the following section (Figure 4 .4A) [314, 315] .

EXPRESSING HETEROLOGOUS SURFACE SUGARS

Glycoengineering methods have found a number of useful applications-the most successful, perhaps, being vaccine design. One effective vaccine platform involves the heterologous expression of carbohydrate antigens (such as glycoproteins [19] , capsular polysaccharides [18,307À309,316À319] , and LPS [311À313,320]) on the surface of attenuated vaccine strains. Analogously to the recombinant protein-expressing vaccines described above, the bacterial vector serves as a potent adjuvant stimulating adaptive immune responses against the pathogen-associated glycoantigen. The second major approach involves attenuation of pathogens through genetic modification of their LPS, allowing direct vaccination with avirulent pathogenic strains. Common molecular alterations to LPS, diagrammed in to produce hexa-acetylated Lipid A (through expression of acyltransferases, LpxL, and LpxM) [314] . In contrast to the immunoevasive native glycoforms, re-engineered LPS variants potently activate toll-like receptor 4 (TLR4), leading to the elicitation of pro-inflammatory immune responses that mediate rapid clearance of the bacteria, and generation of adaptive immunity against the bacterial vector [315] . This method has been applied to construct attenuated vaccines for Shigella flexneri [324] , Y. pestis [21, 325, 326] , Salmonella spp. [327, 328] , Francisella tularensis [20, 329] , Leptospira interrogans [330] , Neisseria meningitides [331] , Moraxella catarrhalis [332] , and K. pneumoniae [333] .

Surface glycoengineering has also been used to develop a potentially revolutionary class of anti-infective probiotics: avirulent bacterial strains expressing oligosaccharide receptors for the bacterial exotoxins that cause cholera, shigellosis, and traveller's diarrhea, which together claim hundreds of thousands of lives per year worldwide [22,334À338] . The rationale for this approach is that exotoxins are sequestered by receptor decoys on probiotic strains in the gut lumen rather than binding to their targets on colonic epithelial cells, which induces diarrheal disease. Due to the density and multivalency of receptor expression on the surface of probiotic strains, exotoxins are intercepted in vivo with extraordinary efficiency. In one study, 1 mg of recombinant bacteria was shown to neutralize over 150 μg of shiga toxin, a potent exotoxin released by Shigella spp. that leads to dysentery. In mice, twice daily administration of the therapeutic strain provided 100% protection against otherwise fatal shigellosis, even in killed form. Thus, in addition to being cost effective (given that large-scale production can be achieved through simple fermentation), this therapeutic approach is highly effective and safe. Furthermore, these re-engineered probiotics apply no selective pressure for evolution of resistant pathogenic strains because they specifically intercept disease-causing toxins rather than killing the pathogen itself. Recently, similar principles have guided the development of a probiotic expressing the glycan receptor for adhesion proteins on uropathogenic strains of E. coli. Prophylactic treatment with the recombinant strain abrogated binding of the pathogen to the urinary epithelium, preventing development of urinary tract infection in mice [339] . Unfortunately, despite the great promise such glycoengineered probiotics hold for the treatment of infectious disease, they have been slowed in their clinical development due to apprehension surrounding the release of GMOs (see Box 4.1).

The final application of glycoengineering relates to production of recombinant glycoproteins. The importance of this process is highlighted by the fact that approximately 70% of therapeutically relevant proteins have saccharide modifications, including catabolic enzymes used for treating inborn errors of metabolism and erythropoietin, which is used in the treatment of chronic kidney disease [340] . These carbohydrate moieties affect several pharmacological properties of glycoproteins, including in vivo activity, half-life, proteolytic stability, and tissue targeting [341À346] . Glycoproteins are also used as vaccines; in fact, some of the most commonly used vaccines-including those against H. influenzae, N. meningitidis, and S. pneumoniae-consist of capsular polysaccharideÀprotein conjugates. Conventional methods for producing such compounds involve isolation of polysaccharide capsule from pathogens, purification of recombinant carrier proteins, chemical coupling of polysaccharides to carrier proteins, and purification of glycoconjugates-a typically low yielding and expensive process [15] . Alternatively, researchers have pursued total synthesis of glycans, but despite having led 

While the strategies presented in this chapter for expressing heterologous proteins and sugars at the bacterial cell surface have proven useful in a myriad of applications, they nevertheless fall short in one important regard: they offer no way of installing non-genetically encoded molecules such as fluorophores, affinity tags, and cytotoxic drugs on bacteria. For such compounds, an alternative set of approaches, pioneered by chemical biologists over the last decade, must be employed.

In general, there are two basic strategies for installing small molecules onto bacterial surfaces: direct and two-step labeling. In direct labeling, small moleculeÀmonomer conjugates are incubated with actively growing cells so that cells take up the conjugate and incorporate it via endogenous biosynthetic pathways into polymeric structures on the cell surface (such as proteins or sugars). In contrast, two-step labeling involves (i) incorporation of a monomer equipped with a small, reactive chemical handle (such as an azide) into a cell surface structure, and (ii) subsequent bioorthogonal ligation of a small molecule functionalized with a complementary reactive group (an alkyne in this example) to the surface-exposed handle. This two-step approach is particularly useful when installing large compounds that are unable to pass the plasma membrane, or too bulky to allow direct incorporation by the enzymes involved with polymer biosynthesis. Besides the azide/terminal alkyne click reaction pair, commonly used bioorthogonal reaction pairs include: azides and phosphines (Staudinger ligation), aldehydes/ketones and hydrazines or hydroxylamines, and tetrazines and strained alkenes such as trans-cyclooctenes [349, 350] . Two-step metabolic labeling of glycans, initially developed by Bertozzi et al. [351] , represents the paradigmatic example of chemical surface engineering. In this methodology, monosaccharides modified with chemical handles are first assimilated into surface-associated sugars and then small molecules of interest are attached to the functionalized glycan at the cell surface. The outer membrane glycolipid, LPS, has served as the most common target for modification [352À354], although recent studies have shown that glycoproteins are amenable to chemical manipulation as well [355À357] .

Polypeptides at the cell surface have been similarly re-engineered via two-step metabolic labeling techniques. In analogy to the functionalized monosaccharides used to label glycans, chemically tagged noncanonical amino acids (ncAAs) are incorporated into proteins and presented on the cell surface for subsequent ligation. To this end, ncAAs such as azidohomoalanine, azidonorvaline, and azidonorleucine have been shown to serve as methionine surrogates, replacing the endogenous amino acid during translation [358À362]. Alkynefunctionalized compounds, in turn, are attached to these protein-bound, surface-exposed azides. Through this approach, methionines are globally replaced with ncAAs-a particularly valuable feature in certain settings (e.g., proteomic analysis). However, for site-specific modification, an alternative technique-pioneered by Schultz et al.-must be used. First, a genetically engineered tRNAÀtRNA synthetase pair, designed to incorporate a ketone-containing to elegant chemical developments [347, 348] , these approaches have been hampered by the extreme chemical complexity of oligosaccharides, deriving from the stereoisomeric diversity of monosaccharide subunits, numerous possible glycosidic linkages, and frequent branching.

Heterologous expression of N-linked glycosylation systems in E. coli, meanwhile, allows for scalable, inexpensive production of easily purifiable, homogeneous glycoconjugates, with site-specific installation of glycans on target proteins. While still in need of improvements (such as identification of bacterial oligosaccharyltransferases that can attach glycans with non-reducing sugars to acceptor proteins), bacterial glycotagging technology has the potential to revolutionize the production of therapeutic and immunomodulatory glycoproteins in the future [16, 17] .

Owing to the self-regenerating nature of the bacterial vector, its immunogenic properties, strong genotypeÀphenotype linkage, and amenability to genetic manipulation, bacterial surface display has proven an extraordinarily versatile and economical biotechnological system. Applications of these strategies in vaccinology have led to agents in clinical trials, while catalytic "biofactories" have played a key role in industrial and pharmaceutical synthesis, and bacteria-based screening technologies have driven forward basic research. However, we believe the surface engineering paradigm may be further improved by combining heterologous expression of protein and saccharides with the many complementary technologies elucidated above including computational protein engineering. Even further, genetic surface display can benefit from integration with an exciting set of methods emerging in chemical biology that allow installation of synthetic compounds on the cell surface (see Box 4.3) [364, 365] . Together, these powerful techniques would impart incredible versatility to the surface engineering platform, transforming the cell envelope into a veritable tabula rasa, able to accommodate all manner of protein, sugar, and synthetic chemical in virtually any combination. With these tools, for instance, one may imagine constructing an anticancer therapeutic strain that expresses (i) a tumor antigen to trigger adaptive immune responses; (ii) an oligosaccharide targeting moiety to promote uptake by APCs and efficient antigen presentation (e.g., the DC-SIGN ligand, Sialyl-Lewis X ) [366À368]; and (iii) a chemotherapeutic payload that is released upon arrival in the tumor microenvironment [225] . Thus, through interdisciplinary efforts involving synthetic chemists, bacteriologists, and biomedical engineers, we imagine the development of a new class of "synthetic organisms"-chemically and genetically re-engineered bacteria that will serve as novel multimodal therapeutics, biotechnological devices, and tools to advance fundamental research.

We deeply appreciate the contributions of Dr. Thihan Padukkavidana toward the completion of this manuscript. ncAA (m-acetyl-L-phenylalanine) into a recombinant surface protein in response to the amber nonsense stop codon, is introduced into a bacterial strain. Then, the strain is incubated with m-acetyl-L-phenylalanine, so that the ncAA is selectively incorporated into the recombinant protein. Finally, the surface-exposed ketone functionality is used as an attachment point for a small molecule containing a hydrazine or hydroxylamine group [363] .

Human immunodeficiency virus

The sensitivity of bacteria to the action of penicillin

The pneumococcal capsular swelling reaction, studied with the aid of the electron microscope

Cell surface exposure of the outer membrane protein OmpA of Escherichia coli K-12

Probing the topology of a bacterial membrane protein by genetic insertion of a foreign epitope; expression at the cell surface

Molecular design of the microbial cell surface toward the recovery of metal ions

Versatile microbial surface-display for environmental remediation and biofuels production

The autodisplay story, from discovery to biotechnical and biomedical applications

Fluorescent and bioluminescent cell-based sensors: strategies for their preservation

Protein engineering with bacterial display

Bioengineered microbes in disease therapy

Biotechnological applications for surface-engineered bacteria

Bacterial spores as platforms for bioanalytical and biomedical applications

Decorating microbes: surface display of proteins on Escherichia coli

Mucosal vaccination and therapy with genetically modified lactic acid bacteria

Recent developments in bacterial protein glycan coupling technology and glycoconjugate vaccine design

Expanding the glycoengineering toolbox: the rise of bacterial N-linked protein glycosylation

Glycansby-design: engineering bacteria for the biosynthesis of complex glycans and glycoconjugates

Intranasal immunization with heterologously expressed polysaccharide protects against multiple Pseudomonas aeruginosa infections

Exploiting the Campylobacter jejuni protein glycosylation system for glycoengineering vaccines and diagnostic tools directed against brucellosis

Attenuated virulence of a Francisella mutant lacking the lipid A 4'-phosphatase

Virulence factors of Yersinia pestis are overcome by a strong lipopolysaccharide response

Bioengineered bugs expressing oligosaccharide receptor mimics: toxinbinding probiotics for treatment and prevention of enteric infections

Biological safety concepts of genetically modified live bacterial vaccines

Live bacterial vaccines-a review and identification of potential hazards

Plasmid maintenance systems suitable for GMO-based bacterial vaccines

Curtiss 3rd R. Cloning and characterization of the asd gene of Salmonella typhimurium: use in stable maintenance of recombinant plasmids in Salmonella vaccine strains

Stabilization of recombinant avirulent vaccine strains in vivo

Recombineering: genetic engineering in bacteria using homologous recombination

High efficiency recombineering in lactic acid bacteria

The bacterial cell envelope

Versatility of a vector for expressing foreign polypeptides at the surface of gram-negative bacteria

Fimbrial surface display systems in bacteria: from vaccines to random libraries

Autodisplay of enzymesmolecular basis and perspectives

Autodisplay of streptavidin

Autodisplay of nitrilase from Klebsiella pneumoniae and wholecell degradation of oxynil herbicides and related compounds

Autodisplay of active sorbitol dehydrogenase (SDH) yields a whole cell biocatalyst for the synthesis of rare sugars

The structures and functions of ice crystal-controlling proteins from bacteria

Surface display of heme-and diflavin-containing cytochrome P450 BM3 in Escherichia coli: a whole cell biocatalyst for oxidation

Extracellular transport of cholera toxin B subunit using Neisseria IgA protease beta-domain: conformation-dependent outer membrane translocation

Surface display of Zymomonas mobilis levansucrase by using the icenucleation protein of Pseudomonas syringae

Functional display of active bovine adrenodoxin on the surface of E. coli by chemical incorporation of the [2FeÀ2S] cluster

Microbial cell-surface display

Display of proteins on bacteria

Anchoring of proteins to lactic acid bacteria

Sortases and the art of anchoring proteins to the envelopes of gram-positive bacteria

Display of alpha-amylase on the surface of Lactobacillus casei cells by use of the PgsA anchor protein, and production of lactic acid from starch

Construction of a functional S-layer fusion protein comprising an immunoglobulin G-binding domain for development of specific adsorbents for extracorporeal blood purification

S-layermediated display of the immunoglobulin G-binding domain of streptococcal protein G on the surface of Caulobacter crescentus: development of an immunoactive reagent

Cell surface display of chimeric glycoproteins via the S-layer of Paenibacillus alvei

S-layer fusion proteins-construction principles and applications

Are S-layers exoskeletons? The basic function of protein surface layers revisited

Bacterial peptidoglycan (murein) hydrolases

System using tandem repeats of the cA peptidoglycan-binding domain from Lactococcus lactis for display of both N-and C-terminal fusions on cell surfaces of lactic acid bacteria

Novel surface display system for proteins on non-genetically modified gram-positive bacteria

Cell surface display system for Lactococcus lactis: a novel development for oral vaccine

Characterization of a novel LysM domain from Lactobacillus fermentum bacteriophage endolysin and its use as an anchor to display heterologous proteins on the surfaces of lactic acid bacteria

Revival and identification of bacterial spores in 25-to 40-million-year-old Dominican amber

Localization of GerAA and GerAC germination proteins in the Bacillus subtilis spore

Display of proteins on Bacillus subtilis endospores

Targeting of the BclA and BclB proteins to the Bacillus anthracis spore surface

Surface display of recombinant proteins on Bacillus thuringiensis spores

Integration of spore-based genetically engineered whole-cell sensing systems into portable centrifugal microfluidic platforms

Packaging sensing cells in spores for long-term preservation of sensors: a tool for biomedical and environmental analysis

Construction of spores for portable bacterial whole-cell biosensing systems

Sposensor: a whole-bacterial biosensor that uses immobilized Bacillus subtilis spores and a one-step incubation/ detection process

Application of a real-time biosensor to detect bacteria in platelet concentrates

Dissecting interactions between nucleosides and germination receptors in Bacillus cereus 569 spores

Immobilized bacterial spores for use as bioindicators in the validation of thermal sterilization processes

Transgalactosylation in a water-solvent biphasic reaction system with betagalactosidase displayed on the surfaces of Bacillus subtilis spores

Oral vaccine delivery by recombinant spore probiotics

Bacillus spores for vaccine delivery

Structure, assembly, and function of the spore surface layers

Spore-displayed streptavidin: a live diagnostic tool in biotechnology

Enhanced immunisation and expression strategies using bacterial spores as heat-stable vaccine delivery vehicles

The intestinal life cycle of Bacillus subtilis and close relatives

Biological functions and biogenesis of secreted bacterial outer membrane vesicles

The Bacterial Ghost platform system: production and applications

Virulence and immunomodulatory roles of bacterial outer membrane vesicles

Engineered bacterial outer membrane vesicles with enhanced functionality

Meningococcal vaccines

Gram-negative outer membrane vesicles in vaccine development

Immunogenicity and safety of a strainspecific MenB OMV vaccine delivered to under 5-year olds in New Zealand

Gramnegative outer membrane vesicles: beyond the cell surface

Bacterial ghosts (BGs)-advanced antigen and drug delivery system

Bacterial ghosts as adjuvant particles

Progress in understanding adjuvant immunotoxicity mechanisms

Beyond pattern recognition: five immune checkpoints for scaling the microbial threat

Protective immunity elicited by recombinant bacille Calmette-Guerin (BCG) expressing outer surface protein A (OspA) lipoprotein: a candidate Lyme disease vaccine

Construction and immunogenicity of recombinant Mycobacterium bovis BCG expressing GP5 and M protein of porcine reproductive respiratory syndrome virus

Immune responses dependent on antigen location in recombinant attenuated Salmonella typhimurium vaccines following oral immunization

Impact of intracellular location of and antigen display by intracellular bacteria: implications for vaccine development

Superior efficacy of secreted over somatic antigen display in recombinant Salmonella vaccine induced protection against listeriosis

An inducible surface presentation system improves cellular immunity against human papillomavirus type 16 E7 antigen in mice after nasal administration with recombinant lactococci

Expression of the Yersinia pestis capsular antigen (F1 antigen) on the surface of an aroA mutant of Salmonella typhimurium induces high levels of protection against plague

Factors affecting the immunogenicity of tetanus toxin fragment C expressed in Lactococcus lactis

Comparison of the immune responses induced by local immunizations with recombinant Lactobacillus plantarum producing tetanus toxin fragment C in different cellular locations

Ability of Lactococcus lactis to export viral capsid antigens: a crucial step for development of live vaccines

Surface display compared to periplasmic expression of a malarial antigen in Salmonella typhimurium and its implications for immunogenicity

Mucosal and systemic immune responses to chimeric fimbriae expressed by Salmonella enterica serovar Typhimurium vaccine strains

Toll-dependent selection of microbial antigens for presentation by dendritic cells

Vaccine delivery: a matter of size, geometry, kinetics and molecular patterns

Salmonella enterica serovar Typhi live vector vaccines finally come of age

Rational design of Salmonella recombinant vaccines

A fusogenic peptide expressed on the surface of Salmonella enterica elicits CTL responses to a dengue virus epitope

Surface-displayed viral antigens on Salmonella carrier vaccine

Surface display of domain III of Japanese encephalitis virus E protein on Salmonella typhimurium by using an ice nucleation protein

Bacterial expression of the major antigenic regions of porcine rotavirus VP7 induces a neutralizing immune response in mice

Salmonella expressing a T-cell epitope from Sendai virus are able to induce anti-infection immunity

Construction, characterization, and immunogenicity of an attenuated Salmonella enterica serovar Typhimurium pgtE vaccine expressing fimbriae with integrated viral epitopes from the spiC promoter

Comparison of a fimbrial versus an autotransporter display system for viral epitopes on an attenuated Salmonella vaccine vector

Towards a human oral vaccine for anthrax: the utility of a Salmonella Typhi Ty21a-based prime-boost immunization strategy

Oral administration of a Salmonella enterica-based vaccine expressing Bacillus anthracis protective antigen confers protection against aerosolized B. anthracis

Oral immunization with a Salmonella typhimurium vaccine vector expressing recombinant enterotoxigenic Escherichia coli K99 fimbriae elicits elevated antibody titers for protective immunity

Expression of recombinant enterotoxigenic Escherichia coli colonization factor antigen I by Salmonella typhimurium elicits a biphasic T helper cell response

Autodisplay: efficacious surface exposure of antigenic UreA fragments from Helicobacter pylori in Salmonella vaccine strains

Protection against murine listeriosis by oral vaccination with recombinant Salmonella expressing protective listerial epitopes within a surface-exposed loop of the TolC-protein

Curtiss 3rd R. A live recombinant avirulent oral Salmonella vaccine expressing pneumococcal surface protein A induces protective responses against Streptococcus pneumoniae

Curtiss 3rd R. PspA family fusion proteins delivered by attenuated Salmonella enterica serovar Typhimurium extend and enhance protection against Streptococcus pneumoniae

Autodisplay: development of an efficacious system for surface display of antigenic determinants in Salmonella vaccine strains

A comparison of immunogenicity and protective immunity against experimental plague by intranasal and/or combined with oral immunization of mice with attenuated Salmonella serovar Typhimurium expressing secreted Yersinia pestis F1 and V antigen

A Salmonella enterica serovar Typhi vaccine expressing Yersinia pestis F1 antigen on its surface provides protection against plague in mice

Enhanced immunity to Plasmodium falciparum circumsporozoite protein (PfCSP) by using Salmonella enterica serovar Typhi expressing PfCSP and a PfCSP-encoding DNA vaccine in a heterologous prime-boost strategy

Expression of the Plasmodium falciparum immunodominant epitope (NANP)(4) on the surface of Salmonella enterica using the autotransporter MisL

Display and release of the Plasmodium falciparum circumsporozoite protein using the autotransporter MisL of Salmonella enterica

Expression of a Porphyromonas gingivalis hemagglutinin on the surface of a Salmonella vaccine vector

Trichinella spiralis: intranasal immunization with attenuated Salmonella enterica carrying a gp43 antigen-derived 30mer epitope elicits protection in BALB/c mice

Attenuated Bordetella pertussis BPZE1 as a live vehicle for heterologous vaccine antigens delivery through the nasal route

Highly attenuated Bordetella pertussis strain BPZE1 as a potential live vehicle for delivery of heterologous vaccine candidates

Development of live attenuated Bordetella pertussis strains expressing the universal influenza vaccine candidate M2e

Production of nontypeable Haemophilus influenzae HtrA by recombinant Bordetella pertussis with the use of filamentous hemagglutinin as a carrier

Production of Neisseria meningitidis transferrin-binding protein B by recombinant Bordetella pertussis

Induction of mucosal immune responses against a heterologous antigen fused to filamentous hemagglutinin after intranasal immunization with recombinant Bordetella pertussis

Development of a tripartite vector system for live oral immunization using a gram-negative probiotic carrier

Delivery of heterologous protein antigens via hemolysin or autotransporter systems by an attenuated ler mutant of rabbit enteropathogenic Escherichia coli

Over-expression of major colonization factors of enterotoxigenic Escherichia coli, alone or together, on non-toxigenic E. coli bacteria

Split immune response after oral vaccination of mice with recombinant Escherichia coli Nissle 1917 expressing fimbrial adhesin K88

Surface display of Salmonella epitopes in Escherichia coli and Staphylococcus carnosus

Construction of a novel Shigella live-vector strain co-expressing CS3 and LTB/STm of enterotoxigenic E. coli

Genetic characterization and immunogenicity of coli surface antigen 4 from enterotoxigenic Escherichia coli when it is expressed in a Shigella live-vector strain

Immunogenicity of multivalent Shigella-ETEC candidate vaccine strains in a guinea pig model

Construction and characterization of bivalent Shigella flexneri 2a vaccine strains SC608 (pCFAI) and SC608(pCFAI/LTB) that express antigens from enterotoxigenic Escherichia coli

Attenuated Shigella flexneri 2a vaccine strain CVD 1204 expressing colonization factor antigen I and mutant heat-labile enterotoxin of enterotoxigenic Escherichia coli

Exploiting cholera vaccines as a versatile antigen delivery platform

Development of a live oral attaching and effacing Escherichia coli vaccine candidate using Vibrio cholerae CVD 103-HgR as antigen vector

Construction of non-toxic Escherichia coli and Vibrio cholerae strains expressing high and immunogenic levels of enterotoxigenic E. coli colonization factor I fimbriae

Mucosal delivery of therapeutic and prophylactic molecules using lactic acid bacteria

Lactococcus lactis as a live vector: heterologous protein production and DNA delivery systems

Development of vaccine delivery vehicles based on lactic acid bacteria

Targeting diseases with genetically engineered Lactococcus lactis and its course towards medical translation

Immunogenicity and protective efficacy of orally administered recombinant Lactococcus lactis expressing surface-bound HIV Env

Vaginal immunization of Cynomolgus monkeys with Streptococcus gordonii expressing HIV-1 and HPV 16 antigens

Oral administration of human papillomavirus type 16 E7 displayed on Lactobacillus casei induces E7-specific antitumor effects in C57/BL6 mice

Bermú dez-Humarán LG. Influence of the route of immunization and the nature of the bacterial vector on immunogenicity of mucosal vaccines based on lactic acid bacteria

A novel mucosal vaccine based on live Lactococci expressing E7 antigen and IL-12 induces systemic and mucosal immune responses and protects mice against human papillomavirus type 16-induced tumors

Commensal bacteria as vectors for mucosal vaccines against sexually transmitted diseases: vaginal colonization with recombinant streptococci induces local and systemic antibodies in mice

Immunization of mice by oral colonization with live recombinant commensal streptococci

Inhibition of HIV infectivity by a natural human isolate of Lactobacillus jensenii engineered to express functional two-domain CD4

Evaluation of oral immunization with recombinant avian influenza virus HA1 displayed on the Lactococcus lactis surface and combined with the mucosal adjuvant cholera toxin subunit B

Oral immunization with live Lactococcus lactis expressing rotavirus VP8 subunit induces specific immune response in mice

Rotavirus vp7 antigen produced by Lactococcus lactis induces neutralizing antibodies in mice

Recombinant porcine rotavirus VP4 and VP4-LTB expressed in Lactobacillus casei induced mucosal and systemic antibody responses in mice

Oral vaccination with the porcine rotavirus VP4 outer capsid protein expressed by Lactococcus lactis induces specific antibody production

Mucosal immunization with surface-displayed severe acute respiratory syndrome coronavirus spike protein on Lactobacillus casei induces neutralizing antibodies in mice

Oral immunization with recombinant Lactobacillus plantarum induces a protective immune response in mice with Lyme disease

Oral vaccination of mice against tetanus with recombinant Lactococcus lactis

Induction of immune responses in mice after oral immunization with recombinant Lactobacillus casei strains expressing enterotoxigenic Escherichia coli F41 fimbrial protein

Immunogenicity and protective efficacy of orally or intranasally administered recombinant Lactobacillus casei expressing ETEC K99

Secretory expression of K88 (F4) fimbrial adhesin FaeG by recombinant Lactococcus lactis for oral vaccination and its protective immune response in mice

Lactobacillus acidophilus expressing recombinant K99 adhesive fimbriae has an inhibitory effect on adhesion of enterotoxigenic Escherichia coli

Oral vaccination of mice against Helicobacter pylori with recombinant Lactococcus lactis expressing urease subunit B

Intranasal immunization of mice with recombinant Streptococcus gordonii expressing NadA of Neisseria meningitidis induces systemic REFERENCES bactericidal antibodies and local IgA

Intranasal immunisation with recombinant Lactococcus lactis displaying either anchored or secreted forms of Proteus mirabilis MrpA fimbrial protein confers specific immune response and induces a significant reduction of kidney bacterial colonisation in mice

Intragastric immunization with recombinant Lactobacillus casei expressing flagellar antigen confers antibody-independent protective immunity against Salmonella enterica serovar Enteritidis

Innate and acquired immune responses induced by recombinant Lactobacillus casei displaying flagellin-fusion antigen on the cell surface

Dissimilar properties of two recombinant Lactobacillus acidophilus strains displaying Salmonella FliC with different anchoring motifs

Use of Lactococcus lactis expressing pili from group B Streptococcus as a broad-coverage vaccine against streptococcal disease

Oral immunization with recombinant Streptococcus lactis carrying the Streptococcus mutans surface protein antigen gene

Mucosal delivery of a pneumococcal vaccine using Lactococcus lactis affords protection against respiratory infection

Resistance of young mice to pneumococcal infection can be improved by oral vaccination with recombinant Lactococcus lactis

Nasal immunization with Lactococcus lactis expressing the pneumococcal protective protein A induces protective immunity in mice

Nasal immunization of mice with Lactobacillus casei expressing the Pneumococcal surface protein A: induction of antibodies, complement deposition and partial protection against Streptococcus pneumoniae challenge

Nasal immunization of mice with Lactobacillus casei expressing the pneumococcal surface protein C primes the immune system and decreases pneumococcal nasopharyngeal colonization in mice

Oral immunization with recombinant Lactococcus lactis confers protection against respiratory pneumococcal infection

Mucosal vaccine made from live, recombinant Lactococcus lactis protects mice against pharyngeal infection with Streptococcus pyogenes

Protection against Yersinia pseudotuberculosis infection conferred by a Lactococcus lactis mucosal delivery vector secreting LcrV

Expression of the Giardia lamblia cyst wall protein 2 in Lactococcus lactis

Immunogenicity of a malaria parasite antigen displayed by Lactococcus lactis in oral immunisations

Mucosal immunisation of mice with malaria protein on lactic acid bacterial cell walls

Age-dependent systemic antibody responses and immunisation-associated changes in mice orally and nasally immunised with Lactococcus lactis expressing a malaria parasite protein

Engineering of staphylococcal surfaces for biotechnological applications

Partial protection to respiratory syncytial virus (RSV) elicited in mice by intranasal immunization using live staphylococci with surface-displayed RSV-peptides

A surface-displayed cholera toxin B peptide improves antibody responses using food-grade staphylococci for mucosal subunit vaccine delivery

Cell-surface display of heterologous epitopes on Staphylococcus xylosus as a potential delivery system for oral vaccination

Immunization against anthrax using Bacillus subtilis spores expressing the anthrax protective antigen

The detection of protective antigen (PA) associated with spores of Bacillus anthracis and the effects of anti-PA antibodies on spore germination and macrophage interactions

Mucosal delivery of antigens using adsorption to bacterial spores

Surface display of recombinant proteins on Bacillus subtilis spores

Display of heterologous antigens on the Bacillus subtilis spore coat using CotC as a fusion partner

Immunogenicity of recombinant Bacillus subtilis spores expressing Clonorchis sinensis tegumental protein

Oral administration of a Bacillus subtilis spore-based vaccine expressing Clonorchis sinensis tegumental protein 22.3 kDa confers protection against Clonorchis sinensis

Immunogenicity of self-adjuvanticity oral vaccine candidate based on use of Bacillus subtilis spore displaying Schistosoma japonicum 26 KDa GST protein

Immunization with Salmonella enterica serovar Typhimurium-derived outer membrane vesicles delivering the pneumococcal protein PspA confers protection against challenge with Streptococcus pneumoniae

OmpA-FMDV VP1 fusion proteins: production, cell surface exposure and immune responses to the major antigenic domain of foot-and-mouth disease virus

Comparative immunogenicity of the hepatitis B virus core 149 antigen displayed on the inner and outer membrane of bacterial ghosts

Bacterial ghosts: non-living candidate vaccines

Recombinant Vibrio cholerae ghosts as a delivery vehicle for vaccinating against Chlamydia trachomatis

A novel recombinant multisubunit vaccine against Chlamydia

Meningococcal outer membrane vesicle vaccines derived from mutant strains engineered to express factor H binding proteins from antigenic variant groups 1 and 2

Protective antibody responses elicited by a meningococcal outer membrane vesicle vaccine with overexpressed genome-derived neisserial antigen 1870

Improved immunogenicity of a H44/76 group B outer membrane vesicle vaccine with over-expressed genomederived Neisserial antigen 1870

Mucosal immunization: a realistic alternative

PAMPs and alarmins: all we need to know about danger

Capsule enhances pneumococcal colonization by limiting mucus-mediated clearance

Glycoprotein 2 (GP2): grabbing the FimH bacteria into M cells for mucosal immunity

M cell-targeted mucosal vaccine strategies

Genetic engineering of Streptococcus gordonii for the simultaneous display of two heterologous proteins at the bacterial surface

The use of clostridial spores for cancer treatment

The treatment of inoperable sarcoma by bacterial toxins (the mixed toxins of the streptococcus erysipelas and the bacillus prodigiosus)

Intracavitary Bacillus Calmette-Guerin in the treatment of superficial bladder tumors

Biodistribution and genetic stability of the novel antitumor agent VNP20009, a genetically modified strain of Salmonella typhimurium

Colonisation of clostridium in the body is restricted to hypoxic and necrotic areas of tumours

Sparse initial entrapment of systemically injected Salmonella typhimurium leads to heterogeneous accumulation within tumors

Salmonella typhimurium specifically chemotax and proliferate in heterogeneous tumor tissue in vitro

Engineering the perfect (bacterial) cancer therapy

Use of preferentially replicating bacteria for the treatment of cancer

Environmentally controlled invasion of cancer cells by engineered bacteria

Short hairpin RNAexpressing bacteria elicit RNA interference in mammals

Inhibition of tumor growth using salmonella expressing Fas ligand

Inhibition of tumor growth and metastasis by a combination of Escherichia colimediated cytolytic therapy and radiotherapy

Genetically engineered Salmonella typhimurium as an imageable therapeutic probe for cancer

Potent antitumor activity of systemically-administered IL2-expressing Salmonella correlates with decreased angiogenesis and enhanced tumor apoptosis

Tumour-targeted delivery of TRAIL using Salmonella typhimurium enhances breast cancer survival in mice

Salmonella promoters preferentially activated inside tumors

Pilot trial of genetically modified, attenuated Salmonella expressing the E. coli cytosine deaminase gene in refractory cancer patients

Phase I study of the intravenous administration of attenuated Salmonella typhimurium to patients with metastatic melanoma

Live, attenuated strains of Listeria and Salmonella as vaccine vectors in cancer treatment

Therapeutic live vaccines as a potential anticancer strategy

Bacterial vectors for active immunotherapy reach clinical and industrial stages

Listeria monocytogenes and its products as agents for cancer immunotherapy

Salmonella enterica as a vaccine carrier

Cancer immunotherapy based on killing of Salmonella-infected tumor cells

Translating tumor antigens into cancer vaccines

The first clinical use of a live-attenuated Listeria monocytogenes vaccine: a Phase I safety study of Lm-LLO-E7 in patients with advanced carcinoma of the cervix

Oral vaccination with attenuated Salmonella enterica strains encoding T-cell epitopes from tumor antigen NY-ESO-1 induces specific cytotoxic T-lymphocyte responses

Mice immunization with live lactococci displaying a surface anchored HPV-16 E7 oncoprotein

A novel mucosal vaccine based on live Lactococci expressing E7 antigen and IL-12 induces systemic and mucosal immune responses and protects mice against human papillomavirus type 16-induced tumors

An inducible surface presentation system improves cellular immunity against human papillomavirus type 16 E7 antigen in mice after nasal administration with recombinant lactococci

Cell wall anchoring of the 37-kilodalton oncofetal antigen by Lactobacillus plantarum for mucosal cancer vaccine delivery

Improving tumor targeting and therapeutic potential of Salmonella VNP20009 by displaying cell surface CEA-specific antibodies

Engineering the third wave of biocatalysis

New opportunities for biocatalysis: making pharmaceutical processes greener

Chemoenzymatic synthesis of small molecule human therapeutics

Recent trends in biocatalysis engineering

Enzymes and bioconversions of industrial, pharmaceutical, and biotechnological significance

PolymerÀenzyme conjugates can self-assemble at oil/water interfaces and effect interfacial biotransformations

Specific adhesion to cellulose and hydrolysis of organophosphate nerve agents by a genetically engineered Escherichia coli strain with a surface-expressed cellulose-binding domain and organophosphorus hydrolase

Autodisplay of functional CYP106A2 in Escherichia coli

Surface display of transglucosidase on Escherichia coli by using the ice nucleation protein of Xanthomonas campestris and its application in glucosylation of hydroquinone

Extracellular transport of VirG protein in Shigella

Functional display of pseudomonas and Burkholderia lipases using a translocator domain of EstA autotransporter on the cell surface of Escherichia coli

Esterase autodisplay: enzyme engineering and whole-cell activity determination in microplates with pH sensors

Genetically engineered whole-cell sensing systems: coupling biological recognition with reporter genes

Serendipitous discovery of two highly selective inhibitors of bacterial luciferase

Sensing antimonite and arsenite at the subattomole level with genetically engineered bioluminescent bacteria

Microbial whole-cell sensing systems of environmental pollutants

A tripartite microbial reporter gene system for real-time assays of soil nutrient status

Biosensing systems for the detection of bacterial quorum signaling molecules

Whole cell strategies based on lux genes for high throughput applications toward new antimicrobials

A quantitative immunoassay utilizing Escherichia coli cells REFERENCES possessing surface-expressed single chain Fv molecules

Epitope mapping and affinity purification of monospecific antibodies by Escherichia coli cell surface display of gene-derived random peptide libraries

Computational design of ligandbinding proteins with high affinity and selectivity

Phage display: concept, innovations, applications and future

Selecting high-affinity binding proteins by monovalent phage display

Epitope mapping using gram-positive surface display

Epitope mapping of antibodies using bacterial surface display

Identification of a new prostatespecific cyclic peptide with the bacterial FliTrx system

Screening and identification of a novel target specific for hepatoma cell line HepG2 from the FliTrx bacterial peptide library

A novel affinity protein selection system based on staphylococcal cell surface display and flow cytometry

Bacterial display enables efficient and quantitative peptide affinity maturation

Exploiting bacterial peptide display technology to engineer biomaterials for neural stem cell culture

Rapid isolation of high-affinity protein binding peptides using bacterial display

Selection of peptide entry motifs by bacterial surface display

Expression of random peptide fused to invasin on bacterial cell surface for selection of cell-targeting peptides

Bacterial display in combinatorial protein engineering

Screening of molecular repertoires by microbial surface display

Peptide discovery using bacterial display and flow cytometry

Bacterial surface display library screening by target enzyme labeling: identification of new human cathepsin G inhibitors

Sequence requirements of the GPNG beta-turn of the Ecballium elaterium trypsin inhibitor II explored by combinatorial library screening

Ultrahigh-throughput FACSbased screening for directed enzyme evolution

Engineering of recombinant antibody fragments to methamphetamine by anchored periplasmic expression

Anchored periplasmic expression, a versatile technology for the isolation of high-affinity antibodies from Escherichia coliexpressed libraries

Protection against anthrax toxin by recombinant antibody fragments correlates with antigen affinity

Isolation of engineered, full-length antibodies from libraries expressed in Escherichia coli

Function-based isolation of novel enzymes from a large library

Engineering of protease variants exhibiting high catalytic activity and exquisite substrate selectivity

An engineered protease that cleaves specifically after sulfated tyrosine

Highly active and selective endopeptidases with programmed substrate specificities

Single-cell high-throughput screening to identify enantioselective hydrolytic enzymes

High-throughput screening methodology for the directed evolution of glycosyltransferases

Fluorescence activated cell sorting as a general ultra-high-throughput screening method for directed evolution of glycosyltransferases

Chemical tools to discover and target bacterial glycoproteins

Protein secretion and surface display in gram-positive bacteria

Capsules of Streptococcus pneumoniae and other bacteria: paradigms for polysaccharide biosynthesis and regulation

Biosynthesis and assembly of capsular polysaccharides in Escherichia coli

Lipopolysaccharide: biosynthetic pathway and structure modification

Oral vaccination of BALB/c mice with Salmonella enterica serovar Typhimurium expressing Pseudomonas aeruginosa O antigen promotes increased survival in an acute fatal pneumonia model

Heterologous expression of an immunogenic pneumococcal type 3 capsular polysaccharide in Lactococcus lactis

Heterologous expression of the pneumococcal serotype 14 polysaccharide in Lactococcus lactis requires lactococcal epsABC regulatory genes

Molecular cloning and characterization of the genetic determinants that express the complete Shigella serotype D (Shigella sonnei) lipopolysaccharide in heterologous live attenuated vaccine strains

Molecular cloning and characterization of genes for Shigella sonnei form I O polysaccharide: proposed biosynthetic pathway and stable expression in a live salmonella vaccine vector

Expression of Shigella dysenteriae serotype 1 O-antigenic polysaccharide by Shigella flexneri aroD vaccine candidates and different S. flexneri serotypes

Construction of a multivalent vaccine strain of Shigella flexneri and evaluation of serotype-specific immunity

Lipid A modification systems in gram-negative bacteria

Fortifying the barrier: the impact of lipid A remodelling on bacterial pathogenesis

Enhanced immunogenicity in the murine airway mucosa with an attenuated Salmonella live vaccine expressing OprFÀOprI from Pseudomonas aeruginosa

Expression of the Streptococcus pneumoniae type 3 synthase in Escherichia coli. Assembly of type 3 polysaccharide on a lipid primer

Genetic exchange of determinants for capsular polysaccharide biosynthesis between Klebsiella pneumoniae strains expressing serotypes K2 and K21a

Manno(rhamno)biosecontaining capsular polysaccharides of Klebsiella pneumoniae enhance opsono-stimulation of human polymorphonuclear leukocytes

Core-linked LPS expression of Shigella dysenteriae serotype 1 O-antigen in live Salmonella Typhi vaccine vector Ty21a: preclinical evidence of immunogenicity and protection

Phosphoethanolamine substitution in the lipid A of REFERENCES Escherichia coli O157:H7 and its association with PmrC

Characterization of the two-protein complex in Escherichia coli responsible for lipopolysaccharide assembly at the outer membrane

Putting endotoxin to work for us: monophosphoryl lipid A as a safe and effective vaccine adjuvant

Two msbB genes encoding maximal acylation of lipid A are required for invasive Shigella flexneri to mediate inflammatory rupture and destruction of the intestinal epithelium

Curtiss 3rd R. A live attenuated strain of Yersinia pestis KIM as a vaccine against plague

A Yersinia pestis lpxM-mutant live vaccine induces enhanced immunity against bubonic plague in mice and guinea pigs

Phosphate groups of lipid A are essential for Salmonella enterica serovar Typhimurium virulence and affect innate and adaptive immunity

Genetic modification of the Opolysaccharide of Francisella tularensis results in an avirulent live attenuated vaccine

Cross-protective immunity against leptospirosis elicited by a live, attenuated lipopolysaccharide mutant

Nextgeneration outer membrane vesicle vaccines against Neisseria meningitidis based on nontoxic LPS mutants

Moraxella catarrhalis bacterium without endotoxin, a potential vaccine candidate

Secondary acylation of Klebsiella pneumoniae lipopolysaccharide contributes to sensitivity to antibacterial peptides

A recombinant probiotic for treatment and prevention of cholera

Recombinant probiotics for treatment and prevention of enterotoxigenic Escherichia coli diarrhea

A new biological agent for treatment of Shiga toxigenic Escherichia coli infections and dysentery in humans

Oral administration of formaldehyde-killed recombinant bacteria expressing a mimic of the Shiga toxin receptor protects mice from fatal challenge with Shiga-toxigenic Escherichia coli

Refinement of a therapeutic Shiga toxin-binding probiotic for human trials

Escherichia coli 83972 expressing a P. fimbriae oligosaccharide receptor mimic impairs adhesion of uropathogenic E. coli

Challenges in therapeutic glycoprotein production

Glycoengineering: the effect of glycosylation on the properties of therapeutic proteins

Effects of glycosylation on the stability of protein pharmaceuticals

Glycosylation of therapeutic proteins: an effective strategy to optimize efficacy

Glycoengineering the N-acyl side chain of sialic acid of human erythropoietin affects its resistance to sialidase

Optimizing the use of erythropoietic agentsÀpharmacokinetic and pharmacodynamic considerations

A comparison of the pharmacological properties of carbohydrate remodeled recombinant and placental-derived beta-glucocerebrosidase: implications for clinical efficacy in treatment of Gaucher disease

Opportunities and challenges in synthetic oligosaccharide and glycoconjugate research

Chemical synthesis of carbohydrates and their surface immobilization: a brief introduction

Chemistry in living systems

Click chemistry: diverse chemical function from a few good reactions

Engineering chemical reactivity on cell surfaces through oligosaccharide biosynthesis

Remodeling bacterial polysaccharides by metabolic pathway engineering

Click-mediated labeling of bacterial membranes through metabolic modification of the lipopolysaccharide inner core

Metabolic incorporation of unnatural sialic acids into Haemophilus ducreyi lipooligosaccharides

Metabolic labeling of fucosylated glycoproteins in Bacteroidales species

Metabolic profiling of Helicobacter pylori glycosylation

The engineering of bacteria bearing azidopseudaminic acid-modified flagella

Discovery of Escherichia coli methionyl-tRNA synthetase mutants for efficient labeling of proteins with azidonorleucine in vivo

Noncanonical amino acids in the interrogation of cellular protein synthesis

Cell surface labeling of Escherichia coli via copper(I)-catalyzed [3 1 2] cycloaddition

Discovery of aminoacyl-tRNA synthetase activity through cell-surface display of noncanonical amino acids

Presentation and detection of azide functionality in bacterial cell surface proteins

A new strategy for the site-specific modification of proteins in vivo

Exterior design: strategies for redecorating the bacterial surface with small molecules

A biosynthetic strategy for re-engineering the Staphylococcus aureus cell wall with non-native small molecules

Exploiting bacterial glycosylation machineries for the synthesis of a Lewis antigencontaining glycoprotein

Targeting C-type lectin receptors with multivalent carbohydrate ligands

Glycan-based DC-SIGN targeting vaccines to enhance antigen cross-presentation