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

Gastrointestinal disease caused by Shiga toxin-producing bacteria (such as Escherichia coli O157:H7 and Shigella dysenteriae) is often complicated by life-threatening toxin-induced systemic sequelae, including hemolytic-uremic syndrome. Such infections can now be diagnosed very early in the course of the disease, but at present no effective therapeutic intervention is possible. Here, we constructed a recombinant bacterium that displayed a Shiga toxin receptor mimic on its surface, and it adsorbed and neutralized Shiga toxins with very high efficiency. Moreover, oral administration of the recombinant bacterium completely protected mice from challenge with an otherwise 100%-fatal dose of Shiga toxigenic E. coli. Thus, the bacterium shows great promise as a 'probiotic' treatment for Shiga toxigenic E. coli infections and dysentery.

The interferon stimulated gene viperin, restricts Shigella. flexneri in vitro.

The role of interferon and interferon stimulated genes (ISG) in limiting bacterial infection is controversial, and the role of individual ISGs in the control of the bacterial life-cycle is limited. Viperin, is a broad acting anti-viral ISGs, which restricts multiple viral pathogens with diverse mechanisms. Viperin is upregulated early in some bacterial infections, and using the intracellular bacterial pathogen, S. flexneri, we have shown for the first time that viperin inhibits the intracellular bacterial life cycle. S. flexneri replication in cultured cells induced a predominantly type I interferon response, with an early increase in viperin expression. Ectopic expression of viperin limited S. flexneri cellular numbers by as much as 80% at 5hrs post invasion, with similar results also obtained for the intracellular pathogen, Listeria monocytogenes. Analysis of viperins functional domains required for anti-bacterial activity revealed the importance of both viperin's N-terminal, and its radical SAM enzymatic function. Live imaging of S. flexneri revealed impeded entry into viperin expressing cells, which corresponded to a loss of cellular cholesterol. This data further defines viperin's multi-functional role, to include the ability to limit intracellular bacteria; and highlights the role of ISGs and the type I IFN response in the control of bacterial pathogens.

Anuran olfactory bulb organization: embryology, neurochemistry and hodology.

In Xenopus laevis, we analyzed the origin of the projection neurons and interneurons in the developing olfactory bulbs by studying the expression patterns of the genes x-Eomes, x-Lhx5, x-Dll3 and x-Pax6. Olfactory bulb interneurons were characterized by using four conserved molecular markers for distinct subpopulations: gamma-aminobutyric acid, calretinin, calbindin, and tyrosine hydroxylase. Immunohistochemistry was combined with tract-tracing experiments to demonstrate the projection neurons and the interneurons of the olfactory bulbs simultaneously. Taken together, the results showed: (1) the pallial nature of the olfactory bulb and its projection neurons in Xenopus, like in mammals with comparable central projection areas, (2) the subpallial origin of the interneurons that, most likely, follow migratory pathways comparable to those described for mammals, (3) the different interneuron types possess neurochemical characteristics similar to mammals. Therefore, the present results show that the origin, chemoarchitecture and central connections of the olfactory bulbs are highly conserved in evolution.

Calbindin-D28k and calretinin as markers of retinal neurons in the anuran amphibian Rana perezi.

In the present study we have analyzed the distribution of the calcium binding proteins calbindin-D28k (CB) and calretinin (CR) immunoreactive cells in the retina of the anuran Rana perezi using poly- and mono-clonal antibodies that were proven to be specific in the amphibian brain, without cross-reactivity. Double immunohistofluorescence techniques were used to demonstrate colocalization of both proteins in the same retinal cells. In addition, retrograde tracing experiments from the optic nerve were conducted to labeled ganglion cells and these were observed in combination with CB and/or CR immunohistochemistry. Cells containing CB were identified as all cones, scattered bipolar and amacrine cells together with cells in the ganglion cell layer. The pattern of CR immunoreactivity was strikingly different. Abundant cells contained CR in the inner retinal layers including horizontal, bipolar and amacrine cells, and cells in the ganglion cell layer. By means of double immunohistochemistry it was found that only subpopulations of amacrine cells and cells in the ganglion cell layer contained both CB and CR. Tracing from the optic nerve revealed retrogradely labeled ganglion cells with different morphologies and most of them contained CB and/or CR. All these data taken together suggest that in amphibians CB and CR are distinctly and precisely distributed in retinal neurons showing, however, peculiar features not observed previously in other vertebrates.

Immunohistochemical localization of neuropeptide FF-like in the brain of the turtle: relation to catecholaminergic structures.

A previous study in the lizard Gekko gecko has revealed that neuropeptide FF (NPFF, a neuropeptide involved in nociception, cardiovascular regulation, and endocrine function) is widely distributed throughout the brain and spinal cord. Although the distribution of NPFF immunoreactivity shares many features with that found in other vertebrates, it was noted that Gekko shared more features with anamniotes in terms of number of cell groups, more elaborate networks of fibers, and lack of colocalization with catecholamines, than with mammals. To assess the primitive or derived character of these features, NPFF and tyrosine hydroxylase (TH) antibodies have been applied to the brain and spinal cord of the turtle, Pseudemys scripta elegans, which belongs to a different radiation of reptiles. As in Gekko, major NPFF-ir cell groups were found in the diagonal band nucleus of Broca and in the hypothalamus, whereas additional cells were identified in the anterior olfactory nucleus, lateral and dorsal cortices, dorsal ventricular ridge, and the intergeniculate leaflet formation. Notable differences are the presence of NPFF-ir cells in the medial cortex and striatum of Pseudemys, which are lacking in Gekko. On the other hand, no NPFF-ir cells could be detected in the septal region and dorsal horn of the spinal cord in Pseudemys. Double staining with NPFF and TH antibodies revealed an intimate relationship between NPFF-ir and TH-ir structures but colocalization could not be established. In conclusion, the distribution of NPFF in the brain of Pseudemys has corroborated previous results in Gekko, but also revealed some notable species differences.

Genetic analysis of the rfbX gene of Shigella flexneri.

Analysis of the nucleotide sequence of the rfbX gene of Shigella flexneri revealed that it contained a high proportion of rare codons, as previously observed in the analysis of the O-antigen polymerase-encoding gene rfc [Morona et al., J. Bacteriol. 176 (1994) 733-747]. The rfbX gene encodes a hydrophobic, 46-kDa protein, with 12 potential transmembrane-spanning domains, that shows structural homology with gene products encoded in many rfb regions, and with Orf0416 of the rff region of Escherichia coli K-12 which has also been identified as a member of this class of proteins. Attempts to clone rfbX independent of other rfb genes, and to identify the protein product of rfbX have proven unsuccessful. Analysis of plasmids containing various deletions within the rfb region suggest that the 5' end of rfbX plays an indirect regulatory role in expression of the dTDP-rhamnose biosynthetic enzymes, encoded by rfbBCAD. We speculate that RfbX is a cytoplasmic membrane protein which functions in the transport of the O-antigen repeat unit.

In Vibrio cholerae serogroup O1, rfaD is closely linked to the rfb operon.

The rfaD gene of Escherichia coli encodes ADP-L-glycero-D-mannoheptose-6- epimerase, an enzyme required for the biosynthesis of the lipopolysaccharide (LPS) precursor ADP-L-glycero-D- mannoheptose, associated with production of the core oligosaccharide. We have identified an rfaD homologue in Vibrio cholerae O1. This gene maps adjacent to the rfb region encoding O-antigen biosynthesis, but is transcribed divergently. The complete nucleotide sequence of rfaD and the flanking DNA has been determined, and rfaD would appear to be the only gene homologous to known LPS core biosynthesis genes in this region. Comparison with the E. coli rfaD shows many similar structural features such as the ADP-binding beta alpha beta fold at the N terminus, as well as a high degree of homology of both the nucleotide and amino-acid sequences. Based on homology, rfaD of V. cholerae may be transcribed using both sigma 70- and sigma 54-dependent promoters.

Putative O-antigen transport genes within the rfb region of Vibrio cholerae O1 are homologous to those for capsule transport.

The nucleotide sequence of that part of the Vibrio cholerae (Vc) O1 rfb region encompassing rfbG, rfbH and rfbI is presented. Expression of these genes has enabled the products for rfbG and rfbI to be confirmed, but the rfbH product has not been detected. Comparisons with the sequences of known proteins reveals that RfbH and RfbI are likely to be involved in the export of lipopolysaccharide (LPS). RfbH shows considerable homology to a number of integral membrane proteins, some of which have been identified as possibly having a role as an export channel for capsular polysaccharides. RfbI corresponds to an ATP-binding protein usually found linked to the membrane protein and is thought to be required for energizing this export process. Thus, we propose that RfbH and RfbI form a complex for the export of Vc O1 LPS. The function of RfbG is unknown, but it would appear to be a relatively hydrophilic protein and we can only speculate that it may be either a specific transferase or possibly the O-antigen polymerase.

Construction of plasmid vectors with a non-antibiotic selection system based on the Escherichia coli thyA+ gene: application to cholera vaccine development.

The construction of live oral carriers based on attenuated Salmonella strains as vectors offers a new approach to vaccine development. We have constructed a set of plasmid vectors which have the thyA gene of Escherichia coli (encoding thymidylate synthetase) as the marker for selection and maintenance of plasmid clones. The thyA system offers an alternative to antibiotic-resistance selection markers. It can be easily adapted to a particular host-vector combination since thyA chromosomal mutations can be readily introduced by trimethoprim selection. We also describe the construction of thyA-based plasmids with the Vibrio cholerae rfb genes (encoding O-antigen biosynthesis of the Inaba serotype). These have been found to be useful in the construction of candidate bivalent cholera-typhoid vaccines.

A putative pathway for biosynthesis of the O-antigen component, 3-deoxy-L-glycero-tetronic acid, based on the sequence of the Vibrio cholerae O1 rfb region.

The nucleotide sequence of a region of the rfb genes, encoding biosynthesis of the Vibrio cholerae (Vc) O1 O-antigen, was determined. Analysis of the open reading frames (ORFs) within this region has revealed similarities with a number of different classes of biosynthetic proteins and enzymes. The ORFs have been designated RfbK, RfbL, RfbM, RfbN and RfbO. RfbK is a small, acidic protein which has similarity to the family of proteins known as acyl-carrier proteins (ACP). The RfbL protein has similarity to a super-family of enzymes which adenylate their substrates as a part of their reaction mechanism. Included in these are several acetyl-CoA ligases. Alignment of RfbL with these proteins reveals a highly conserved domain containing the motif GlyXaaXaaGlyXaaPro. This resembles the ATP-binding site motif and may represent a variant of the usual motif, except that Pro replaces Gly. The VcRfbM protein has similarity with a family of long-chain, iron-containing alcohol dehydrogenases, of which the Escherichia coli K-12 fucO and adhE gene products are also members. The RfbN protein has sequence homology with LuxE and LuxC of Vibrio harveyi (Vh) and other bioluminescent bacterial species. The latter are two components of the enzyme complex which synthesizes the long-chain aldehyde used in the V. harveyi bioluminescence system. Finally, the VcRfbO protein has sequence similarity with acetyl-CoA transferases. We were able to identify a number of the gene products using a T7 expression system, confirming several of the allocated ORFs. A biosynthetic pathway for the Vc O-antigen component 3-deoxy-L-glycero-tetronic acid, based on the enzymatic functions predicted for the RfbK, RfbL, RfbM, RfbN and RfbO proteins, is presented.

A putative pathway for perosamine biosynthesis is the first function encoded within the rfb region of Vibrio cholerae O1.

The first four genes (rfbA,B,D,E) of the rfb region of Vibrio cholerae O1 are predicted to encode the enzymes required for the biosynthesis of perosamine, which constitutes the backbone structure of the O-antigen of the lipopolysaccharide. Based on homology to known proteins/protein families, the following functions are predicted: RfbA, phosphomannose isomerase-guanosine diphosphomannose pyrophosphorylase; RfbB, phosphomanno-mutase; RfbD, oxido reductase and RfbE, perosamine synthetase (amino-transferase). Thus, perosamine is synthesized from fructose 6-phosphate via the intermediates mannose 6-phosphate by RfbA, to mannose 1-phosphate by RfbB, to GDP-mannose by RfbA, to GDP-4-keto-6-dideoxymannose by RfbD and to GDP-perosamine by RfbE. This final product would then serve as the substrate for the addition of the tetronate, which could then be polymerized into the O-antigen for transfer to the lipid A plus core oligosaccharide and export to the cell surface. The organization of these genes are such that one would expect them to be translationally coupled as part of the rfb operon. However, the absence of readily detectable promoter sequences suggests low levels of transcription, in line with other studies. The nucleotide sequence of these genes is absolutely conserved in the two isolates 569B (classical, Inaba) and O17 (El Tor, Ogawa) which were expected to show maximal sequence variation. This suggests very tight constraints on the micro-evolution within these sequences.

A physical map of the chromosomal region determining O-antigen biosynthesis in Vibrio cholerae O1.

We have previously described the cosmid cloning of the genes determining the biosynthesis of the Inaba and Ogawa O-antigens of the lipopolysaccharides of Vibrio cholerae O1 (Manning et al., 1986). By Southern hybridization analysis of chromosomal and cosmid DNA, and heteroduplex analysis between the clones we have been able to precisely define the region of contiguous chromosomal DNA in the vicinity of the O-antigen-encoding region. These data and comparison of end points of clones and of deletion derivatives demonstrate that at least 16 kb of a 19-kb SstI fragment is required to encode O-antigen biosynthesis. Expression of O-antigen is independent of the orientation of this SstI fragment with respect to cloning vectors suggesting that its regulatory region has been cloned intact. No detectable differences were observed in the restriction patterns of the Inaba and Ogawa coding regions implying that only minor changes are involved when serotype conversion (Inaba to Ogawa or vice versa) occurs. Bhaskaran [Ind. J. Med. Res. 47 (1959) 253-260] originally defined this region associated with O-antigen biosynthesis oag; however, to be consistent with other organisms [Hitchcock et al., J. Bacteriol. 166 (1986) 699-705], it is suggested this be changed to rfb.

Characterization of the capsular polysaccharide biosynthesis locus of Streptococcus pneumoniae type 19F.

We have used a combination of plasmid insertion/rescue and inverse Polymerase Chain Reaction (PCR) to clone the region of the Streptococcus pneumoniae type 19F chromosome encoding biosynthesis of type 19F capsular polysaccharide (cps19f), which was then subjected to sequence analysis. The cps19f locus is located in the S. pneumoniae chromosome between dexB and aliA, and consists of 15 open reading frames (ORFs), designated cps19fA to cps19fO, that appear to be arranged as a single transcriptional unit. Insertion-duplication mutants in 13 of the 15 ORFs have been constructed in a smooth type 19F strain, all of which resulted in a rough (unencapsulated) phenotype, confirming that the operon is essential for capsule production. Comparison with sequence databases has allowed us to propose functions for 12 of the cps19f gene products, and a biosynthetic pathway for type 19F capsular polysaccharide. Southern hybridization analysis indicated that cps19fA and cps19fB were the only cps genes found in all 16 S. pneumoniae serotypes/groups tested. The region from cps19fG to cps19fK was found only in members of serogroup 19, and within this cps19fI was unique to type 19F.

Effect of lipopolysaccharide core synthesis mutations on the production of Vibrio cholerae O-antigen in Escherichia coli K-12.

The rfb genes of Vibrio cholerae O1 (Ogawa serotype) were subcloned into a derivative of pBR322. This plasmid was transformed into several Escherichia coli K-12 mutant strains which produce an incomplete lipopolysaccharide (LPS)-core-oligosaccharide region. The data indicate that the V. cholerae O-antigen is assembled onto the E. coli LPS and that at least two glucoses are needed in the core in order to achieve a high level of production. These data are consistent with the reported presence of glucose in the V. cholerae LPS-core-oligosaccharide region.

Differential immunogenicity of Vibrio cholerae O139 variants expressing different combinations of naturally occurring and atypical forms of the serogroup polysaccharide.

Field testing of an inactivated bivalent O1/O139 cholera vaccine suggests that Vibrio cholerae O1 is more immunogenic than V. cholerae O139. To investigate whether this might be partly attributable to the production of capsular polysaccharide (CPS) by O139 isolates, we have compared the immunogenicity of variant strains expressing different combinations of lipopolysaccharide (LPS) and CPS. These studies indicate that the core-linked LPS structure is of paramount importance for induction of antibodies to the serogroup antigen. By contrast CPS was minimally immunogenic. Significantly the presence of CPS did not modulate the immunogenicity of the underlying LPS. To examine whether differences in LPS structure might contribute to the differing immunogenicities of the O1 and O139 serogroups, an attempt was made to modify the normal O139 LPS structure by provision of one of several heterologous wzz genes. The resulting variants displayed additional, atypical surface polysaccharide, whose modal length was characteristic for the particular wzz gene. By immunoblotting this novel material showed a ladder-like banding pattern typical of LPS, but its failure to be stained by silver indicated that it was not core-associated and was therefore more like truncated CPS. Consistent with our earlier findings, studies using systemic or mucosal routes of immunization failed to demonstrate any consistent enhancement of antibody responses associated with production of these aberrant polysaccharide polymers.

Molecular cloning of the tolC locus of Escherichia coli K-12 with the use of transposon Tn10.

We have cloned the tolC gene of E. coli K-12 into pSF2124 by using transposon Tn10 as the marker to first isolate the relevant DNA fragment. The gene is on a 10.5 kb EcoRI fragment, and Tn5 insertion mutagenesis locates the gene near one end of this EcoRI fragment. An EcoRI-PstI fragment has been subcloned into pBR322 to facilitate further analysis of the gene.

New locus (ttr) in Escherichia coli K-12 affecting sensitivity to bacteriophage T2 and growth on oleate as the sole carbon source.

The nature of resistance to phage T2 in Escherichia coli K-12 was investigated by analyzing a known phage T2-resistant mutant and by isolating new T2-resistant mutants. It was found that mutational alterations at two loci, ompF (encoding the outer membrane protein OmpF) and ttr (T-two resistance), are needed to give full resistance to phage T2. A ttr::Tn10 mutation was isolated and was mapped between aroC and dsdA, where the fadL gene (required for long-chain fatty acid transport) is located. The receptor affected by ttr was the major receptor used by phage T2 and was located in the outer membrane. Phage T2 was thus able to use two outer membrane proteins as receptors. All strains having a ttr::Tn10 allele and most of the independently isolated phage T2-resistant mutants were unable to grow on oleate as the sole carbon and energy source, i.e., they had the phenotype of fadL mutants. The gene fadL is known to encode an inner membrane protein. The most likely explanation is that fadL and ttr are in an operon and that ttr encodes an outer membrane protein which functions in translocating long-chain fatty acids across the outer membrane and also as a receptor for phage T2.

The tolC locus of Escherichia coli affects the expression of three major outer membrane proteins.

tolC mutants, which are resistant to colicin E1 and also highly sensitive to detergents and dyes, were shown to lack the OmpF outer membrane protein. There was little effect on transcription as judged by the use of an ompF-lac operon fusion strain, and the tolC effect was probably due to a post-transcriptional effect. The NmpC protein and protein 2 were also tolC dependent.

Analysis of Shigella flexneri wzz (Rol) function by mutagenesis and cross-linking: wzz is able to oligomerize.

The modal length or degree of polymerization (dp) of the Shigella flexneri O-antigen is determined in an unknown manner by the Wzz/Rol protein. The Wzz protein is anchored into the cytoplasmic membrane by two transmembrane domains (TM1 amino acids 32-52; TM2 amino acids 295-315) with the central loop of the protein located in the periplasm. Plasmids were constructed encoding hybrid Wzz proteins consisting of regions of S. flexneri Wzz (WzzSF) and Salmonella typhimurium Wzz (WzzST). These imparted O-antigen modal chain lengths that implied that the carboxy-terminal region of Wzz was involved in chain length determination. Site-directed mutagenesis was undertaken to investigate the functional significance of highly conserved residues in amino-/carboxy-terminal domains of WzzSF. Some of the WzzSF variants resulted in O-antigen modal chain lengths much shorter than those of wild-type WzzSF, whereas other mutants inactivated WzzSF function entirely and a third class had a longer O-antigen chain length distribution. The data indicate that amino acids throughout the length of the WzzSF protein are important in determination of O-antigen modal chain length. In vivo cross-linking experiments were performed to investigate the interactions between Wzz proteins. The experiments indicated that the WzzSF protein is able to form dimers and oligomers of at least six WzzSF proteins. A carboxy-terminal-truncated WzzSF protein having the amino terminal 194 amino acids was able to oligomerize, indicating that the amino-terminal region is sufficient for the Wzz-Wzz interaction observed. Shortened WzzSF proteins having internal deletions in the amino-terminal region were also able to oligomerize, suggesting that residues 59-194 are not essential for oligomerization. Cross-linking of WzzSF proteins with mutationally altered residues showed that loss of WzzSF function may be correlated to a reduced/altered ability to form oligomers, and that mutational alteration of glycine residues in the TM2 segment affects WzzSF-WzzSF dimer mobility in SDS polyacrylamide gels. These results provide the first evidence of protein-protein interactions for proteins involved in O-antigen polysaccharide biosynthesis.

Host range mutants of bacteriophage Ox2 can use two different outer membrane proteins of Escherichia coli K-12 as receptors.

The Escherichia coli K-12 outer membrane protein OmpA functions as the receptor for bacteriophage Ox2. We isolated a host range mutant of this phage which was able to grow on an Ox2-resistant ompA mutant producing an altered OmpA protein. From this mutant, Ox2h5, a second-step host range mutant was recovered which formed turbid plaques on a strain completely lacking the OmpA protein. From one of these mutants, Ox2h10, a third-step host range mutant, Ox2h12, was isolated which formed clear plaques on a strain missing the OmpA protein. Ox2h10 and Ox2h12 apparently were able to use both outer membrane proteins OmpA and OmpC as receptors. Whereas there two proteins are very different with respect to primary structures and functions, the OmpC protein is very closely related to another outer membrane protein, OmpF, which was not recognized by Ox2h10 or Ox2h12. An examination of the OmpC amino acid sequence, in the regions where it differs from that of OmpF, revealed that one region shares considerable homology with a region of the OmpA protein which most likely is required for phage Ox2 receptor activity.