Lipopolysaccharides promote binding and unfolding of the antibacterial colicin E3 rRNAse domain.

Nuclease colicins are antibacterial proteins produced by certain strains of E. coli to reduce competition from rival strains. These colicins are generally organized with an N-terminal transport (T)-domain, a central receptor binding (R)-domain, and a C-terminal cytotoxic nuclease domain. These colicins are always produced in complex with an inhibitory immunity protein, which dissociates prior entrance of the cytotoxic domain in the target cell. How exactly colicins traverse the cell envelope is not understood, yet this knowledge is important for the design of new antibacterial therapies. In this report, we find that the cytotoxic rRNAse domain of colicin E3, lacking both T- and R-domains, is sufficient to inhibit cell growth provided the immunity protein Im3 has been removed. Thus, while the T-domain is needed for dissociation of Im3, the rRNAse alone can associate to the cell surface without R-domain. Accordingly, we find a high affinity interaction (Kd ~1-2µM) between the rRNAse domain and lipopolysaccharides (LPS). Furthermore, we show that binding of ColE3 to LPS destabilizes the secondary structure of the toxin, which is expectedly crucial for transport through the narrow pore of the porin OmpF. The effect of LPS on binding and unfolding of ColE3 may be indicative of a broader role of LPS for transport of colicins in general.

A Chimeric protein of CFA/I, CS6 subunits and LTB/STa toxoid protects immunized mice against enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia Coli (ETEC) strains are the commonest bacteria causing diarrhea in children in developing countries and travelers to these areas. Colonization factors (CFs) and enterotoxins are the main virulence determinants in ETEC pathogenesis. Heterogeneity of CFs is commonly considered the bottleneck to developing an effective vaccine. It is believed that broad spectrum protection against ETEC would be achieved by induced anti-CF and anti-enterotoxin immunity simultaneously. Here, a fusion antigen strategy was used to construct a quadrivalent recombinant protein called 3CL and composed of CfaB, a structural subunit of CFA/I, and CS6 structural subunits, LTB and STa toxoid of ETEC. Its anti-CF and antitoxin immunogenicity was then assessed. To achieve high-level expression, the 3CL gene was synthesized using E. coli codon bias. Female BALB/C mice were immunized with purified recombinant 3CL. Immunized mice developed antibodies that were capable of detecting each recombinant subunit in addition to native CS6 protein and also protected the mice against ETEC challenge. Moreover, sera from immunized mice also neutralized STa toxin in a suckling mouse assay. These results indicate that 3CL can induce anti-CF and neutralizing antitoxin antibodies along with introducing CFA/I as a platform for epitope insertion.

Structural design principles for specific ultra-high affinity interactions between colicins/pyocins and immunity proteins.

The interactions of the antibiotic proteins colicins/pyocins with immunity proteins is a seminal model system for studying protein-protein interactions and specificity. Yet, a precise and quantitative determination of which structural elements and residues determine their binding affinity and specificity is still lacking. Here, we used comparative structure-based energy calculations to map residues that substantially contribute to interactions across native and engineered complexes of colicins/pyocins and immunity proteins. We show that the immunity protein α1-α2 motif is a unique structurally-dissimilar element that restricts interaction specificity towards all colicins/pyocins, in both engineered and native complexes. This motif combines with a diverse and extensive array of electrostatic/polar interactions that enable the exquisite specificity that characterizes these interactions while achieving ultra-high affinity. Surprisingly, the divergence of these contributing colicin residues is reciprocal to residue conservation in immunity proteins. The structurally-dissimilar immunity protein α1-α2 motif is recognized by divergent colicins similarly, while the conserved immunity protein α3 helix interacts with diverse colicin residues. Electrostatics thus plays a key role in setting interaction specificity across all colicins and immunity proteins. Our analysis and resulting residue-level maps illuminate the molecular basis for these protein-protein interactions, with implications for drug development and rational engineering of these interfaces.

The membrane channel-forming colicin A: synthesis, secretion, structure, action and immunity.

The study of colicin release from producing cells has revealed a novel mechanism of secretion. Instead of a built-in 'tag', such as a signal peptide containing information for secretion, the mechanism employs coordinate expression of a small protein which causes an increase in the envelope permeability, resulting in the release of the colicin as well as other proteins. On the other hand, the mechanism of entry of colicins into sensitive cells involves the same three stages of protein translocation that have been demonstrated for various cellular organelles. They first interact with receptors located at the surface of the outer membrane and are then transferred across the cell envelope in a process that requires energy and depends upon accessory proteins (TolA, TolB, TolC, TolQ, TolR) which might play a role similar to that of the secretory apparatus of eukaryotic and prokaryotic cells. At this point, the type of colicin described in this review interacts specifically with the inner membrane to form an ion channel. The pore-forming colicins are isolated as soluble proteins and yet insert spontaneously into lipid bilayers. The three-dimensional structures of some of these colicins should soon become available and site-directed mutagenesis studies have now provided a large number of modified polypeptides. Their use in model systems, particularly those in which the role of transmembrane potential can be tested for polypeptide insertion and ionic channel gating, constitutes a powerful handle with which to improve our understanding of the dynamics of protein insertion into and across membranes and the molecular basis of membrane excitability. In addition, their immunity proteins, which exist only in one state (membrane-inserted) will also contribute to such an understanding.

Colicin E3 and its immunity genes.

A DNA segment of plasmid ColE3-CA38 was cloned into pBR328 and its nucleotide sequence was determined. This segment contains the putative promoter-operator region, the structural genes of protein A (gene A) and protein B (gene B) of colicin E3, and a part of gene H. Just behind the promoter region, there is an inverted repeat structure of two 'SOS boxes', the specific binding site of the lexA protein. This suggests that the expression of colicin E3 is regulated directly by the lexA protein. Genes A and B face the same direction, with an intergenic space of nine nucleotides between them. ColE3-CA38 and ColE1-K30 are homologous in their promoter-operator regions, but hardly any homology was found in their structural genes. On the other hand, ColE3-CA38 is fairly homologous to CloDF13 throughout the regions sequenced, with some exceptions including putative receptor-binding regions. By deletion mapping of the immunity gene and recloning of gene B, it was shown genetically that protein B itself is the actual immunity substance of colicin E3. It was also found that the expression of E3 immunity partially depends on the recA function. Thus, we propose two modes of expression of E3 immunity: in the uninduced state, only a slight amount of protein B is produced constitutively to protect the cell from being attacked by the exogenous colicin; and in the SOS-induced state, a large amount of protein B is produced to protect the protein synthesis system of the host cell from ribosome inactivation by endogenously produced colicin E3.

A molecular, genetic and immunological approach to the functioning of colicin A, a pore-forming protein.

We have constructed, by recombinant DNA techniques, one hybrid protein, colicin A-beta-lactamase (P24), and two modified colicin As, one (P44) lacking a large central domain and the other (PX-345) with a different C-terminal region. The regulation of synthesis, the release into the medium and the properties of these proteins were studied. Only P44 was released into the medium. This suggests that both ends of the colicin A polypeptide chain might be required for colicin release. None of the three proteins was active on sensitive cells in an assay in vivo. However, P44 was able to form voltage-dependent channels in phospholipid planar bilayers. Its lack of activity in vivo is therefore probably caused by the inability to bind to the receptor in the outer membrane. PX-345 is a colicin in which the last 43 amino acids of colicin A have been replaced by 27 amino acids encoded by another reading frame in the same region of the colicin A structural gene; it was totally unable to form pores in planar bilayers at neutral pH but showed a very slight activity at acidic pH. These results confirm that the C-terminal domain of colicin A is involved in pore formation and indicate that at least the 43 C-terminal amino acid residues of this domain play a significant role in pore formation or pore function. Fifteen monoclonal antibodies directed against colicin A have been isolated by using conventional techniques. Five out of the 15 monoclonal antibodies could preferentially recognize wild-type colicin A. In addition, the altered forms of the colicin A polypeptide were used to map the epitopes of ten monoclonal antibodies reacting specifically with colicin A. Some of the antibodies did not bind to colicin A when it was pre-incubated at acidic pH suggesting that colicin A undergoes conformational change at about pH 4. The effects of monoclonal antibodies on activity in vivo of colicin A were investigated. The degree of inhibition observed was related to the location of the epitopes, with monoclonal antibodies reacting with the N terminus giving greater inhibition. The monoclonal antibodies directed against the C-terminal region promoted an apparent activation of colicin activity in vivo.

Integration of the colicin A pore-forming domain into the cytoplasmic membrane of Escherichia coli.

The pore-forming domain of colicin A (pfColA) fused to a prokaryotic signal peptide (sp-pfColA) inserted into the inner membrane of Escherichia coli and apparently formed a functional channel, when generated in vivo. We investigated pfColA functional activity in vivo by the PhoA gene fusion approach, combined with cell fractionation and protease susceptibility experiments. Alkaline phosphatase was fused to the carboxy-terminal end of each of the ten alpha-helices of sp-pfColA to form a series of differently sized fusion proteins. We suggest that the alpha-helices anchoring pfColA in the membrane are first translocated into the periplasm. We identify two domains that anchor pfColA to the membrane in vivo: domain 1, extending from helix 1 to helix 8, which contains the voltage-responsive segment and domain 2 consisting of the hydrophobic helices 8 and 9. These two domains function independently. Fusion proteins with a mutation inactivating the voltage-responsive segment or with a domain 1 lacking helix 8 were peripherally associated with the outside of the inner membrane, and were therefore digested by proteases added to spheroplasts. In contrast, fusion proteins with a functional domain 1 were protected from proteases, suggesting as expected that most of domain 1 is inserted into the membrane or is indeed translocated to the cytoplasm during pfColA channel opening.

Pore-forming colicins: synthesis, extracellular release, mode of action, immunity.

Colicins are bacterial toxins encoded by plasmids which also confer immunity to producing cells. In a first stage, colicins are synthesized in the cytoplasm of colicinogenic cells. Subsequently they are released into the extracellular medium following the action of a small protein synthesized coordinately with the colicins. This protein is a lipoprotein and causes a non-specific increase in the envelope permeability, in particular, through the activation of an outer membrane phospholipase. After release into the medium, colicins kill sensitive cells in 3 defined steps: adsorption onto a specific receptor at the surface of the bacterium, translocation across the outer membrane and action. A specific domain of the colicin molecule is responsible for each of these steps. The most common colicins are those which kill by depolarizing the cytoplasmic membrane with the formation of voltage-dependent ionic channels. Immunity is conferred to producing cells by a membrane protein which interacts with the colicin and prevents formation or functioning of these ionic channels formed by its C-terminal domain.

Colicin E1 plasmid as a probe for detection and study of anti-dna activity in SLE sera.

Tritiated colicin E1 plasmid DNA ([3H]DNA) was purified, characterized and developed as a test antigen for study of anti-DNA antibody activity in a modified Farr assay. The homogeneous molecular weight (4.2 X 10(6) daltons), physical state (intact covalently closed circles), and capacity for intrinsic radioactive labeling to a suitable specific activity were all important properties of the plasmid probe. In addition, the ability of the Farr assay to measure primary antigen/antibody interactions made quantitative determinations of anti-double-stranded DNA (dsDNA) antibody activity possible. Sonication of the plasmid resulted in definable fragments which were thermally denatured and used in the measurement of anti-single-stranded DNA (ssDNA) antibody activity. Results from studies in which unlabeled dsDNA, ssDNA, amd free 2'-deoxyribonucleotides were employed to inhibit binding of [3H]DNA by anti-DNA antibodies indicated the presence of 3 distinct anti-DNA specificities in SLE sera.

Dessociation and reconstitution of colicin E3 and immunity substance complex.

It has recently been found that so-called native colicin E3, which has been used for studies of its mode of action, is a complex of two kinds of proteins. The complex could be dissociated into the two components in SDS. These components were isolated by gel filtration of 1% SDS followed by treatment with Sowex-2 to remove bounds SDS. One component, characterized by its low molecular weight, prevented colicin E3-induced inhibition of poly(U)-dependent protein synthesis and was designated as immunity substance. The other component (protein A), which was of high molecular weight, had 100-fold higher in vitro ribosome-inactivating activity than native colicin E3, but had lower bacteriocidal activity. Colicin E3 was reconstituted from the two isolated protein components. The reconstituted colicin E3, when compared with protein A, showed a decrease in in vitro activity (inhibition of poly(U)-dependent protein synthesis), but had higher bacteriocidal activity in vivo. Thus complex formation of protein A with immunity substance should play and important role in the bacteriocidal action, but protein A itself might inactivate ribosomes in the interior of the sensitive cells.

Recognition of the colicin A N-terminal epitope 1C11 in vitro and in vivo in Escherichia coli by its cognate monoclonal antibody.

We demonstrate that the 1C10 monoclonal antibody (mAb) directed against the N-terminal domain of the colicin A recognizes a 13 residue-region (13Thr-Gly-Trp-Ser-Ser-Glu-Arg-Gly-Ser-Gly-Pro- Asp-Pro25). When this peptide is inserted into a protein in the amino-terminal or an internal position, the tagged protein is efficiently detected by the 1C11 mAb either by immunoblotting or immunoprecipitation. In vitro, the minimal structure required for detection using the pepscan system is 19Arg-Gly-Ser-Gly-Pro-Glu-Pro25, indicating that in vivo the proper exposure of the epitope requires additional residues. The construction of a versatile vector allowing overproduction of tagged proteins is described. Various applications of the 1C11 epitope are mentioned. This epitope did not alter the function of any of the proteins so far tested.

Colicins: a minireview.

Colicin are toxins that kill specifically E. coli bacteria. These cells have both an outer and a cytoplasmic membrane. Thus, three steps can be distinguished in colicin action: a) the interaction with specific receptors located at the cell surface, b) the uptake through the outer and eventually through the inner membrane, c) the action of the cell target that leads to cell death. In this short review, an overlook of these three steps is given.

[Organization of the genes responsible for colicin Ib synthesis and immunity to it in plasmid ColIb-P9]. / Izuchenie organizatsii genov, otvetstvennykh za sintez kolitsina Ib i immunitet k nemu, v plazmide ColIb-P9.

To study the localization and expression of the ColIb-P9 plasmid genes responsible for colicin Ib synthesis and immunity to it, we isolated a series of Tn5 insertion mutants of recombinant plasmid pIV41 containing the colicin Ib gene in EcoRI fragment of ColIb-P9 (2.7 kb) and the deletion plasmid carrying only a part of the colicin gene. The direction of colicin Ib gene transcription was determined by the analysis of the polypeptides synthesized in minicells carrying the mutant plasmids. The pIV41 plasmid containing cells have been shown to be resistant to both colicin Ib and Ia activities. This type of resistance is usually associated with chromosomal mutations resulting in loss of cell receptors for colicin Ib adsorption. Apparently, the EcoRI fragment of ColIb-P9 studied contains no gene responsible for immunity to colicin. It has been shown that this gene is a portion of SalI fragment (22 kb) of the ColIb-P9, the fragment also carrying the gene which mediates synthesis of colicin Ib.

[Characteristics of hybrid plasmids carrying the genes of colicinogenic plasmid Collb-P9 responsible for colicin Ib synthesis and inhibition of phage T5 development]. / Kharakteristika gibridnykh plazmid, nesushchikh geny kolitsino-gennoi plazmidy Collb-P9, otvetstvenny za sintez kolitsina Ib i podavlenie razvitiia faga T5.

The EcoRI and HindII restriction endonucleases and pBR325 vector plasmid were used to obtain a set of hybrid plasmids containing ColIb-P9 fragments carrying the characters for colicin Ib synthesis and immunity and the ability to inhibit T5 phage growth. The genes responsible for colicin synthesis and immunity are closely linked and localized in the EcoRI fragment with a molecular weight of 1.85 MD (pIV41) or in the HindII fragment of 2.4 MD (pIV1). The clones containing these plasmids show an increased level of both spontaneous and mitomycin C-induced colicin synthesis and an increased level of immunity due to a larger dosage of the genes. The genes controlling T5 growth inhibition are localized in other restriction fragments of ColIb DNA: the EcoRI fragment of 1.45 MD (pIV7) and the HindII fragment of 4.3 MD (pIV5). We have demonstrated by means of hybrid plasmids that T5 growth inhibition is not connected with the colicin Ib synthesized in infected cells and is controlled by other specific product(s) of the ColIb plasmid genes. T5 phage growth was as efficient in clones containing plasmids with cloned colicin Ib genes as in a strain without plasmids. An investigation of the expression of the genes inhibiting T5 phage growth in an in vitro protein synthesis system has revealed a protein with a molecular weight of 36 000 which seems to take part in the process.

[The immunological activity of the bacterial strain Escherichia coli M17 used in preparing a commercial preparation of colibacterin]. / Immunologicheskaia aktivnost' bakterii shtamma Escherichia coli M17, ispol'zuemogo pri izgotovlenii kommercheskogo preparata kolibakterina.

The toxicity, immunogenic properties and protective activity of the live culture of E. coli M17 and antigenic preparations obtained from cell suspensions of this strain have been studied under experimental conditions. As revealed in experiments on mice, E. coli M17 live culture has low virulence, moderate toxicity and provides the protection of immunized mice from challenge with homologous and highly virulent E. coli strains. E. coli M17 live culture, when introduced orally or intravenously into rabbits, ensures the synthesis of 02 and H6 antibodies. Blood sera taken from immunized rabbits yield better results than initial sera in experiments on the passive protection of mice. The results of our experiments show the expediency of the clinical trials of Colibacterin as a perspective Escherichia live oral vaccine.

Immunity protein release from a cell-bound nuclease colicin complex requires global conformational rearrangement.

Nuclease colicins bind their target receptor BtuB in the outer membrane of sensitive Escherichia coli cells in the form of a high-affinity complex with their cognate immunity proteins. The release of the immunity protein from the colicin complex is a prerequisite for cell entry of the colicin and occurs via a process that is still relatively poorly understood. We have previously shown that an energy input in the form of the cytoplasmic membrane proton motive force is required to promote immunity protein (Im9) release from the colicin E9/Im9 complex and colicin cell entry. We report here that engineering rigidity in the structured part of the colicin translocation domain via the introduction of disulfide bonds prevents immunity protein release from the colicin complex. Reduction of the disulfide bond by the addition of DTT leads to immunity protein release and resumption of activity. Similarly, the introduction of a disulfide bond in the DNase domain previously shown to abolish channel formation in planar bilayers also prevented immunity protein release. Importantly, all disulfide bonds, in the translocation as well as the DNase domain, also abolished the biological activity of the Im9-free colicin E9, the reduction of which led to a resumption of activity. Our results show, for the first time, that conformational flexibility in the structured translocation and DNase domains of a nuclease colicin is essential for immunity protein release, providing further evidence for the hypothesis that global structural rearrangement of the colicin molecule is required for disassembly of this high-affinity toxin-immunity protein complex prior to outer membrane translocation.