Age-specific prevalence of antibody to enterotoxigenic Escherichia coli in Ecuadorian and German children

Serum samples from 1397 Ecuadorian children 0-5 years of age were tested by ELISA for antibodies to enterotoxigenic Escherichia coli (ETEC). A gradual prevalence and titer increase was seen for IgG antibodies to heat-labile enterotoxin in children 6-18 months old; 90% prevalences were reached in the second year of life. At this age less than 10% of West German children showed IgG antibodies to heat-labile enterotoxin. In Ecuador age-related ETEC-specific antibody titer increases correlated with age-related prevalence of diarrheal disease. On the other hand, pooled lipopolysaccharide from different ETEC-associated O serogroups could not be used as a seroepidemiologic marker of ETEC infections.     

Identification of antigens by monoclonal antibody PD4 and its expression in Escherichia coli

AIM: To clone and express the antigen of monoclonal antibody (MAb) PD4 for further investigation of its function. METHODS: MGC803 cDNA expression library was constructed and screened with PD4 as probes to clone the antigen. After failed in the library screening, immunoprecipitation and SDS-polyacrylamide gel electrophoresis were applied to purify the antigen for sequence analysis. The antigen coming from Mycoplasma hyorhinis (M. hyorhinis) was further confirmed with Western blot analysis by infecting M. hyorhinis -free HeLa cells and eliminating the M. hyorhinis from MGC803 cells. The full p37 gene was cloned by PCR and expressed successfully in Escherichia coli after site-directed mutations. Immunofluorescence assay was used to demonstrate if p37 protein could directly bind to gastric tumor cell AGS. RESULTS: The cDNA library constructed with MGC803 cells was screened by MAb PD4 as probes. Unfortunately, the positive clones identified with MAb PD4 were also reacted with unrelated antibodies. Then, immunoprecipitation was performed and the purified antigen was identified to be a membrane protein of Mycoplasma hyorhinis (M. hyorhinis) by sequencing of N-terminal amino acid residues. The membrane protein was intensively verified with Western blot by eliminating M. hyorhinis from MGC803 cells and by infecting M. hyorhinis-free HeLa cells. The full p37 gene was cloned and expressed successfully in Escherichia coli after site-directed mutations. Immunofluorescence demonstrated that p37 protein could directly bind to gastric tumor cell AGS. CONCLUSION: The antigen recognized by MAb PD4 is from M. hyorhinis, which suggests the actions involved in MAb PD4 is possibly mediated by p37 protein or M. hyorhinis. As p37 protein can bind directly to tumor cells, the pathogenic role of p37 involved in tumorigenesis justifies further investigation.     

The cyanobacterial taxis protein HmpF regulates type IV pilus activity in response to light

Motility is ubiquitous in prokaryotic organisms including the photosynthetic cyanobacteria where surface motility powered by type 4 pili (T4P) is common and facilitates phototaxis to seek out favorable light environments. In cyanobacteria, chemotaxis-like systems are known to regulate motility and phototaxis. The characterized phototaxis systems rely on methyl-accepting chemotaxis proteins containing bilin-binding GAF domains capable of directly sensing light, and the mechanism by which they regulate the T4P is largely undefined. In this study we demonstrate that cyanobacteria possess a second, GAF-independent, means of sensing light to regulate motility and provide insight into how a chemotaxis-like system regulates the T4P motors. A combination of genetic, cytological, and protein–protein interaction analyses, along with experiments using the proton ionophore carbonyl cyanide m-chlorophenyl hydrazine, indicate that the Hmp chemotaxis-like system of the model filamentous cyanobacterium Nostoc punctiforme is capable of sensing light indirectly, possibly via alterations in proton motive force, and modulates direct interaction between the cyanobacterial taxis protein HmpF, and Hfq, PilT1, and PilT2 to regulate the T4P motors. Given that the Hmp system is widely conserved in cyanobacteria, and the finding from this study that orthologs of HmpF and T4P proteins from the distantly related model unicellular cyanobacterium Synechocystis sp. strain PCC6803 interact in a similar manner to their N. punctiforme counterparts, it is likely that this represents a ubiquitous means of regulating motility in response to light in cyanobacteria.

Motility is ubiquitous in prokaryotic organisms, including both swimming motility in aqueous environments and twitching or gliding motility on solid surfaces, and enables these organisms to optimize their position in response to various environmental factors. Among the photosynthetic cyanobacteria, surface motility is widespread and facilitates phototaxis to seek out favorable light environments (1, 2), and, for multicellular filamentous cyanobacteria, plays a key role in dispersal as well as the establishment of nitrogen-fixing symbioses with eukaryotes (3) and the formation of supracellular structures (3–5).Current understanding of cyanobacterial surface motility at the molecular level has been informed primarily by studies of two model organisms, the unicellular strain Synechocystis sp. strain PCC6803 (herein Synechocystis) and the filamentous strain Nostoc punctiforme ATCC29133/{"type":"entrez-protein","attrs":{"text":"PCC73102","term_id":"1245706357"}}PCC73102, where motility is exhibited only by differentiated filaments termed “hormogonia.” Motility in both organisms is powered by a type IV pilus (T4P) system where the ATPases PilB and PilT drive the extension and subsequent retraction, respectively, of pili which adhere to the substrate and pull the cells forward (for review, see ref. 6). In Synechocystis, the T4P motors are distributed throughout the entire cell, allowing a 360 ° range of motion (7), whereas in N. punctiforme they are confined to rings at the cell poles (8), resulting in movement only along the long axis of the filament. Comparative genomics implies that this mechanism of motility is widely conserved among cyanobacteria (9).Both Synechocystis and N. punctiforme employ chemotaxis-like systems to regulate motility. One of these systems, the Hmp chemotaxis-like system of N. punctiforme (3, 10), and its orthologous counterpart, the Pil chemotaxis-like system of Synechocystis (11), includes homologs to the canonical Escherichia coli chemotaxis complex (for review, see ref. 12), including the histidine kinase CheA, the adaptor protein CheW, the response regulator CheY, and the methyl-accepting chemotaxis protein MCP. These systems are essential for motility in their respective organisms and appear to regulate the T4P motors, although there are distinct differences in the phenotypes for inactivation of the components from each. In Synechocystis, null mutations either enhance or reduce the level of surface piliation (11), whereas in N. punctiforme they disrupt the coordinated polarity, but not the overall level of piliation, and affect various other aspects of hormogonium development (3, 10). In N. punctiforme, the subcellular localization of this system has been determined and has been found arrayed in static, bipolar rings similar to the T4P motors (3). However, the signals that are perceived by the MCPs and the precise mechanism by which these systems modulate T4P activity is currently undefined.Recently, an additional component of the Hmp system, HmpF, was characterized (9). HmpF is a predicted coiled-coil protein and is ubiquitous to, but confined within, the cyanobacterial lineage (9). It is essential for accumulation of surface pili and exhibits dynamic, unipolar localization to the leading poles of most cells in hormogonium filaments (9). Based on these findings, a model has been proposed where the localization of HmpF is regulated by the other components of the Hmp system, and in turn, the unipolar accumulation of HmpF leads to the activation of the T4P motors on one side of the cell to facilitate directional movement.A second chemotaxis-like system in each organism, the Ptx system of N. punctiforme (13) and the Pix system of Synechocystis (14, 15), is essential for positive phototaxis. These systems contain MCPs with cyanobacteriochrome sensory domains capable of perceiving light (for review, see ref. 16). Disruption of the Pix system results in negative phototaxis under light conditions that normally produce a positive phototactic response (14). Several other proteins containing cyanobacteriochromes, and one containing a BLUF domain, also modulate phototaxis in Synechocystis (for review, see ref. 6). In N. punctiforme, disruption of the Ptx system abolishes the phototactic response completely, resulting in uniform movement in all directions regardless of the light conditions (13), and there are currently no other proteins reported to modulate phototaxis. More recently, a motile, wild isolate of the model unicellular cyanobacterium Synechococcus elongatus sp. PCC7942 was shown to possess a chemotaxis-like system that modulates phototaxis in a manner similar to that of the N. punctiforme Ptx system (17). How these systems influence T4P activity to facilitate phototaxis is also currently unknown.There is also a substantial body of literature on motility and phototaxis in cyanobacteria, primarily based on observational studies of various filamentous strains, that predates the development of genetically tractable model organisms (for review, see ref. 18). These reports suggested that the photosystems may serve a sensory role in modulating phototaxis and that proton motive force (PMF) powers motility (19, 20), a finding that is inconsistent with the theory that cyanobacteria possess a common T4P-based gliding motor driven by ATP hydrolysis. In this study, we help reconcile this historical data with more recent molecular studies by providing evidence that the Hmp chemotaxis-like system senses light, possibly indirectly through alterations in PMF, and in turn modulates the interaction of HmpF with the T4P base to activate the motors.     

Local and systemic antibody responses to naturally acquired enterotoxigenic Escherichia coli diarrhea in an endemic area

Fifteen patients hospitalized with acute, watery diarrhea and with enterotoxigenic Escherichia coli (ETEC) detected from stool samples were studied to evaluate the extent to which natural ETEC diarrhea induces local and systemic antibody responses to E. coli heat-labile toxin (LT), homologous lipopolysaccharide (LPS), and colonization factors (CFA/I and CFA/II). Specific IgA and IgG antibodies to LT, CFA I and II, and each patient's homologous LPS were determined by ELISA in serum, saliva, breastmilk, and intestinal lavage fluid. The majority of patients had greater than a twofold rise in local levels of IgA antibodies in the intestine: 80% of LT+ patients responded to LT, 63% of CFA+ patients responded to CFA, and 78% of all toxin-positive patients responded to the LPS of their infecting strain. Local antibody responses in the intestine were associated with responses in breastmilk and saliva, but relationships were not clear-cut, and the usefulness of these secretions as proxy measures of local intestinal antibody production remains unclear. Antibody responses in serum also occurred in most patients and were significantly more frequent in cases than in controls. This study demonstrates that natural ETEC disease results in local IgA responses to LT, CFA, and LPS in the gut and also in immune responses in breastmilk, saliva, and serum.     

Characterization of the antibody response in mice with type II collagen-induced arthritis, using monoclonal anti-type II collagen antibodies

Twenty monoclonal antibodies reactive with type II collagen were characterized as to their determinant specificity and their reactivity with cartilage-derived components. The monoclonal antibodies reacted with 7 different epitopes on the native type II collagen triple helical structure. Antibodies defining 3 of these epitopes occurred more frequently in sera from arthritic mice than in sera from nonarthritic mice. In vivo injection of some selected autoreactive antibodies caused synovitis, but in no case did it give rise to full-blown arthritis.     

Natural history of enteroaggregative and enterotoxigenic Escherichia coli infection among US travelers to Guadalajara,Mexico

The natural history of enteroaggregative Escherichia coli (EAEC) and enterotoxigenic E. coli (ETEC) infection was studied among 40 US travelers who provided weekly stool samples for 4 weeks after arrival in Mexico. At enrollment, 5 subjects were colonized by EAEC and 3 by ETEC. During the first 2 weeks after enrollment, 12 developed EAEC diarrhea, 7 developed ETEC diarrhea (5 with mixed EAEC/ETEC diarrhea), 13 had EAEC colonization, and 7 had ETEC colonization. During the third and fourth weeks, 4 experienced EAEC diarrhea, 2 experienced ETEC diarrhea (1 with mixed EAEC/ETEC diarrhea), 31 had EAEC colonization, and none had ETEC colonization. Plasmid DNA analysis showed a high degree of heterogeneity among EAEC isolates. Symptomatic EAEC infection occurred early after arrival in Guadalajara, Mexico, and was as common as ETEC infection. Asymptomatic EAEC infection was recurrent.     

Frequencies of lipopolysaccharide core types among clinical isolates of Escherichia coli defined with monoclonal antibodies.

Mouse monoclonal antibodies (MAbs) specific for the lipopolysaccharide (LPS) core types R1, R2, and R3 of Escherichia coli and a cross-reactive MAb that binds to the LPS core of almost all E. coli were used in ELISA to determine the frequency of cores resembling R1, R2, and R3 in strains of E. coli isolated from clinical samples (blood and urine specimens) and from the feces of asymptomatic individuals. Of the 180 wild-type isolates, 123 were assigned to R1 core type, 14 to R2, and 18 to R3. Twenty-five wild-type E. coli isolates could not be assigned to a particular core type and may have either an R4 or K12 core or a previously unrecognized core type. R1 core type was associated with O types 1, 4, 6, 8, and 18 and with K1 or K5 capsules. R3 was associated with O15. O75 isolates could be of either R1 or R2 core type.     

Human monoclonal antibody with protective activity for Escherichia coli K1 and Neisseria meningitidis group B infections

We produced human monoclonal antibody that demonstrated specific reactivity to the K1 capsule of Escherichia coli and the group B polysaccharide of Neisseria meningitidis. The antibody was nonreactive with several strains of K1- E. coli and other gram-negative bacteria. All E. coli K1 clinical isolates tested were reactive with the antibody. When assayed for in vitro opsonophagocytic ability, the antibody caused bacterial removal only in the presence of human complement and neutrophils, an observation suggesting a non-bacteriolytic, neutrophil-dependent killing mechanism. Finally, and perhaps most importantly, the antibody was highly protective for infectious disease when used prophylactically in three animal models. The data suggest a potential use for human monoclonal antibodies in preventing and/or treating infections of the blood.     

Identification by DNA hybridization of enterotoxigenic Escherichia coli in a longitudinal study of villages in Thailand

Radioactively labeled enterotoxin genes were used to study the epidemiology of enterotoxigenic Escherichia coli infections in two Thai villages. When E. coli that were isolated from 674 specimens were fixed on nitrocellulose paper and examined for hybridization with E. coli enterotoxin gene probes in Bangkok, the technique had a sensitivity of 94% (31 of 33) and a specificity of 100% (641 of 641), when compared with tests of E. coli for enterotoxin production in the Y-1-adrenal cell and suckling-mouse assays. However, when the same specimens were fixed directly onto nitrocellulose paper at a field laboratory and transported to the reference laboratory for assay with the gene probes, 27 specimens that contained enterotoxigenic E. coli did not hybridize with the E. coli gene probes. Enterotoxigenic E. coli that hybridized with the LT, ST-H, and ST-P probes were identified in 10% (17 of 177) of villagers with diarrhea, 7% (8 of 108) of contacts of individuals with diarrhea caused by enterotoxigenic E. coli, and 3% (32 of 1,199) of persons not associated with cases of diarrhea caused by enterotoxigenic E. coli. Enterotoxigenic E. coli that hybridized with the ST-II probe was not a cause of diarrhea. Alternative methods of retaining DNA on filters under field conditions are needed before this technique can be used for direct examination of specimens with enterotoxin gene probes.     

Failure of monoclonal antibodies to core glycolipid to bind intact smooth strains of Escherichia coli

To study the ability of antibody to the core glycolipid (CGL) region of lipopolysaccharide (LPS) to bind intact gram-negative bacterial cells, we produced monoclonal antibodies that bind LPS from the Escherichia coli rough mutant J5. Four representative monoclonal antibodies that bound four distinct epitopes on the CGL region of LPS were studied. All four antibodies bound both isolated J5 LPS and intact J5 bacterial cells, but none of the antibodies bound to intact cells of E. coli O111:B4 or K1:O7. Binding of the monoclonal antibodies to isolated LPS from these latter two smooth strains was variable. These results confirm the presence of shared antigenic sites in the CGL region of heterologous LPS molecules but indicate that these sites are not necessarily available on smooth gram-negative bacteria for binding by antibody to CGL.     

Characterization of the antibody response in mice with type II collagen–induced arthritis,using monoclonal anti–type II collagen antibodies

Twenty monoclonal antibodies reactive with type II collagen were characterized as to their determinant specificity and their reactivity with cartilage-derived components. The monoclonal antibodies reacted with 7 different epitopes on the native type II collagen triple helical structure. Antibodies defining 3 of these epitopes occurred more frequently in sera from arthritic mice than in sera from nonarthritic mice. In vivo injection of some selected autoreactive antibodies caused synovitis, but in no case did it give rise to full-blown arthritis.     

Toxins and colonization factor antigens of enterotoxigenic Escherichia coli among residents of Jakarta, Indonesia

Infection caused by enterotoxigenic Escherichia coli (ETEC) poses a serious health problem among children and adults in developing countries. Colonization of the small intestinal mucosa by ETEC strains is mediated by antigenically specific fimbriae, also known as colonization factor antigens (CFA). The significance of this study arises from reports that active and passive immunization with ETEC strains harboring CFAs has previously been shown to induce protective immunity against diarrhea in animal models. The aim of this study was to determine toxin-associated CFAs of ETEC isolated from a diarrheal disease case-control study in Jakarta, Indonesia. Thirteen hundred and twenty-three diarrheic and control patients with lactose-fermenting colonies were screened by ganglioside GM1-enzyme-linked immunosorbent assay (GM1-ELISA) for heat-labile (LT) and heat-stable (ST) toxins. Two hundred and forty-six (19%) ETEC isolates identified by GM1-ELISA for the LT/ST toxins were screened for CFAs by Dot blot assay using monoclonal antibodies against CFA/I, II, and IV and against the putative colonization antigens (PCF) PCFO159, PCFO166, CS7, and CS17. Of the 246 ETEC isolates, 177 (72%) elaborated ST, 56 (23%) produced LT, while 13 (5%) elicited both the ST and LT toxins. CFA testing of the 246 ETEC isolates showed that 21 (8%) expressed CFA/I, 3 (1%) exhibited CFA/II, 14 (6%) elaborated CFA/IV, while 7 (3%) expressed PCFO159 and PCFO159 plus CS5. No CFAs or PCFs could be associated with 201 (82%) of the ETEC strains. This report documents the types of CFAs associated with ETEC strains in Jakarta, Indonesia. These data may help current research efforts on the development of CFA-based vaccines for humans against ETEC and provide additional information for future ETEC vaccine trials in Southeast Asia.     

Chirality sensing by Escherichia coli topoisomerase IV and the mechanism of type II topoisomerases

Escherichia coli topoisomerase (Topo) IV is an essential type II Topo that removes DNA entanglements created during DNA replication. Topo IV relaxes (+) supercoils much faster than (-) supercoils, promoting replication while sparing the essential (-) supercoils. Here, we investigate the mechanism underlying this chiral preference. Using DNA binding assays and a single-molecule DNA braiding system, we show that Topo IV recognizes the chiral crossings imposed by the left-handed superhelix of a (+) supercoiled DNA, rather than global topology, twist deformation, or local writhe. Monte Carlo simulations of braid, supercoil, and catenane configurations demonstrate how a preference for a single-crossing geometry during strand passage can allow Topo IV to perform its physiological functions. Single-enzyme braid relaxation experiments also provide a direct measure of the processivity of the enzyme and offer insight into its mechanochemical cycle.     

Cross-reactivity of monoclonal antibodies to Escherichia coli J5 with heterologous gram-negative bacteria and extracted lipopolysaccharides

A panel of eight murine monoclonal antibodies (MAbs) were produced against heat-killed Escherichia coli J5 and shown to react with J5 lipopolysaccharide (LPS) by enzyme-linked immunosorbent assay (ELISA). These antibodies were then assayed by a suspension ELISA for reactivity with up to 20 heterologous smooth or rough isolates of gram-negative bacteria, which were assayed after heat or formalin treatment, or as live cells. Extracted LPS from the same bacteria were tested for reactivity with the MAbs by direct ELISA. The MAbs demonstrated broad cross-reactivity with most heat-treated bacteria. In contrast, cross-reactivity of the MAbs with live or formalin-treated bacteria was limited almost exclusively to E. coli J5, Hemophilus species, or rough mutants of Salmonella minnesota. Reactivity with extracted LPSs and lipid A varied considerably depending on the MAb. Further, when Western blotting was used as the assay only four of eight MAbs reacted with J5 LPS, and none of the MAbs reacted with LPS from smooth S. minnesota or any of its rough mutants. Adsorption of the MAbs with acid hydrolyzed, boiled, or live E. coli J5 prior to ELISA of the MAbs with J5 LPS supported evidence that none of the MAbs were specific for lipid A and that reactivity was greater with boiled than with live cells. Thus, the cross-reactivity of antibodies to E. coli J5 LPS is dependent on the physical state of the bacteria or LPS used for assay, the assay used, and the specificity of the antibody.     

A comparative study of enterotoxin gene probes and tests for toxin production to detect enterotoxigenic Escherichia coli

Escherichia coli isolated from children with diarrhea were tested for enterotoxin production and for hybridization with gene probes for heat-labile (LT) and heat-stable (ST-H and ST-P) enterotoxin. Fecal specimens were also examined directly for genes coding for enterotoxins. E. coli that hybridized with the cloned enterotoxin gene probes was identified by colony hybridization from 46 children, by enterotoxin production from 38 children, and by specimen hybridization from 37 of 304 children examined. Eighty-six percent (473 of 550) of E. coli that hybridized with the cloned DNA probes produced enterotoxins. Four E. coli that hybridized with the LT and 73 E. coli that hybridized with the ST-H probes were nonenterotoxigenic. These isolates were subsequently shown not to hybridize with other constructions of the same probes and did not hybridize with synthetic single-stranded oligonucleotides directed against the LT or ST genes.