Specificity of the Type II Secretion Systems of Enterotoxigenic Escherichia coli and Vibrio cholerae for Heat-Labile Enterotoxin and Cholera Toxin

The Gram-negative type II secretion (T2S) system is a multiprotein complex mediating the release of virulence factors from a number of pathogens. While an understanding of the function of T2S components is emerging, little is known about what identifies substrates for export. To investigate T2S substrate recognition, we compared mutations affecting the secretion of two highly homologous substrates: heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli (ETEC) and cholera toxin (CT) from Vibrio cholerae. Each toxin consists of one enzymatic A subunit and a ring of five B subunits mediating the toxin''s secretion. Here, we report two mutations in LT''s B subunit (LTB) that reduce its secretion from ETEC without global effects on the toxin. The Q3K mutation reduced levels of secreted LT by half, and as with CT (T. D. Connell, D. J. Metzger, M. Wang, M. G. Jobling, and R. K. Holmes, Infect. Immun. 63:4091-4098, 1995), the E11K mutation impaired LT secretion. Results in vitro and in vivo show that these mutants are not degraded more readily than wild-type LT. The Q3K mutation did not significantly affect CT B subunit (CTB) secretion from V. cholerae, and the E11A mutation altered LT and CTB secretion to various extents, indicating that these toxins are identified as secretion substrates in different ways. The levels of mutant LTB expressed in V. cholerae were low or undetectable, but each CTB mutant expressed and secreted at wild-type levels in ETEC. Therefore, ETEC''s T2S system seems to accommodate mutations in CTB that impair the secretion of LTB. Our results highlight the exquisitely fine-tuned relationship between T2S substrates and their coordinate secretion machineries in different bacterial species.Gram-negative bacteria have evolved a number of methods to secrete proteins into the extracellular milieu, with at least six specific secretion systems currently described (14, 30). Type II secretion (T2S), or the main terminal branch of the general secretory pathway, is a feature of a number of proteobacteria and has been shown to be required for pathogenesis and maintenance of environmental niches in a large number of species (5). The T2S system is a multiprotein complex of 12 to 15 components that spans the inner and outer membranes, allowing for the controlled release of certain folded proteins that have been directed to the periplasm through the Sec or Tat machinery (21). Aside from providing a means of exporting freely released virulence factors from plant, animal, and human pathogens (5), the T2S system has been shown to export surface-associated virulence factors (18), fimbrial components (46), outer membrane cytochromes (36), and a surfactant required for sliding motility in Legionella pneumophila (39), among other substrates.While an increasing number of studies have focused on understanding the structure and function of the components of the T2S system itself, little is known about what identifies a periplasmic protein as a substrate for secretion (21, 32). Because proteins secreted from the same bacterial species need not share any obvious structural homology, it is not even clear how much of a T2S substrate interacts with the secretion machinery (32). Analysis of two similar substrates that can each be secreted by the T2S systems of two distinct species would provide information about species-specific identification of T2S substrates and, by extension, the nature of the “secretion motif” identifying those substrates. Heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli (ETEC) and cholera toxin (CT) from Vibrio cholerae represent one such pair of substrates.ETEC and V. cholerae are enteric pathogens causing significant morbidity and mortality worldwide (33). The causative agents of traveler''s diarrhea and cholera, respectively, these two pathogens share a number of similarities, including the nature of their disease symptoms (38). Each pathogen secretes an AB₅ toxin important for colonization and the induction of water and electrolyte efflux from intestinal epithelial cells (1, 29). These toxins, LT and CT, are both encoded by two-gene operons. After sec-dependent transport to the periplasm, holotoxin formation occurs spontaneously (13), with one catalytic A subunit (LTA or CTA) assembling with five B subunits (LTB or CTB), which are responsible for the binding properties of the toxins. Export of fully folded and assembled LT or CT is then accomplished by the T2S system (34, 40). In ETEC, this system is encoded by gspC to -M (40), while in V. cholerae, these genes are found in the eps operon (34).LT and CT are very similar in structure, sharing approximately 80% sequence homology and 83% identity in the mature B subunit (16, 24). ETEC is thought to have acquired the genes for CT through horizontal transfer, with the toxins evolving over time to possess slight differences (45). As such, these toxins share the same primary host receptor, the monosialoganglioside G_M1, and catalyze the same ADP-ribosylation reaction within host cells (38). However, LT is able to bind other host sphingolipids in addition to G_M1 and to interact with sugar residues from the A-type blood antigen, which CT cannot bind (16, 41). Both LT and CT are able to associate with sugar residues in lipopolysaccharide (LPS) on the surface of E. coli cells (17). Binding to each of these substrates can be impaired by point mutation (26, 43).In this study, we report point mutations impairing the release of LT from ETEC and CT from V. cholerae. We analyzed the specificity of the defects in substrate recognition by comparing the effects of substituting charged and neutral residues in key regions of LTB and CTB. To confirm that the identified mutations resulted specifically in a secretion defect, we tested the effect of the mutations on (i) ligand binding by each toxin, (ii) toxin stability, and (iii) formation of secretion-competent B-subunit pentamers. By introducing comparable mutations into both toxins, including one previously reported to impair the secretion of CT (6), and exchanging toxin substrates between the two species, we have revealed species-dependent differences in T2S substrate recognition. Although wild-type LT and CT can be heterologously expressed and secreted from V. cholerae and ETEC, respectively, the substrate residues identified by the secretion machinery in each species are distinct. Together, our results demonstrate that highly homologous T2S substrates are recognized in different ways when secreted by two distinct systems.     

Evaluating the A-Subunit of the Heat-Labile Toxin (LT) As an Immunogen and a Protective Antigen Against Enterotoxigenic Escherichia coli (ETEC)

Diarrheal illness contributes to malnutrition, stunted growth, impaired cognitive development, and high morbidity rates in children worldwide. Enterotoxigenic Escherichia coli (ETEC) is a major contributor to this diarrheal disease burden. ETEC cause disease in the small intestine by means of colonization factors and by production of a heat-labile enterotoxin (LT) and/or a small non-immunogenic heat-stable enterotoxin (ST). Overall, the majority of ETEC produce both ST and LT. LT induces secretion via an enzymatically active A-subunit (LT-A) and a pentameric, cell-binding B-subunit (LT-B). The importance of anti-LT antibodies has been demonstrated in multiple clinical and epidemiological studies, and a number of potential ETEC vaccine candidates have included LT-B as an important immunogen. However, there is limited information about the potential contribution of LT-A to development of protective immunity. In the current study, we evaluate the immune response against the A-subunit of LT as well as the A-subunit’s potential as a protective antigen when administered alone or in combination with the B-subunit of LT. We evaluated human sera from individuals challenged with a prototypic wild-type ETEC strain as well as sera from individuals living in an ETEC endemic area for the presence of anti-LT, anti-LT-A and anti-LT-B antibodies. In both cases, a significant number of individuals intentionally or endemically infected with ETEC developed antibodies against both LT subunits. In addition, animals immunized with the recombinant proteins developed robust antibody responses that were able to neutralize the enterotoxic and cytotoxic effects of native LT by blocking binding and entry into cells (anti-LT-B) or the intracellular enzymatic activity of the toxin (anti-LT-A). Moreover, antibodies to both LT subunits acted synergistically to neutralize the holotoxin when combined. Taken together, these data support the inclusion of both LT-A and LT-B in prospective vaccines against ETEC.     

Relationship between Heat-Labile Enterotoxin Secretion Capacity and Virulence in Wild Type Porcine-Origin Enterotoxigenic Escherichia coli Strains

Heat-labile enterotoxin (LT) is an important virulence factor secreted by some strains of enterotoxigenic Escherichia coli (ETEC). The prototypic human-origin strain H10407 secretes LT via a type II secretion system (T2SS). We sought to determine the relationship between the capacity to secrete LT and virulence in porcine-origin wild type (WT) ETEC strains. Sixteen WT ETEC strains isolated from cases of severe diarrheal disease were analyzed by GM1ganglioside enzyme-linked immunosorbent assay to measure LT concentrations in culture supernatants. All strains had detectable LT in supernatants by 2 h of culture and 1 strain, which was particularly virulent in gnotobiotic piglets (3030-2), had the highest LT secretion level all porcine-origin WT strains tested (P<0.05). The level of LT secretion (concentration in supernatants at 6-h culture) explained 92% of the variation in time-to-a-moribund-condition (R² = 0.92, P<0.0001) in gnotobiotic piglets inoculated with either strain 3030-2, or an ETEC strain of lesser virulence (2534-86), or a non-enterotoxigenic WT strain (G58-1). All 16 porcine ETEC strains were positive by PCR analysis for the T2SS genes, gspD and gspK, and bioinformatic analysis of 4 porcine-origin strains for which complete genomic sequences were available revealed a T2SS with a high degree of homology to that of H10407. Maximum Likelihood phylogenetic trees constructed using T2SS genes gspC, gspD, gspE and homologs showed that strains 2534-86 and 3030-2 clustered together in the same clade with other porcine-origin ETEC strains in the database, UMNK88 and UMN18. Protein modeling of the ATPase gene (gspE) further revealed a direct relationship between the predicted ATP-binding capacities and LT secretion levels as follows: H10407, -8.8 kcal/mol and 199 ng/ml; 3030-2, -8.6 kcal/mol and 133 ng/ml; and 2534-86, -8.5 kcal/mol and 80 ng/ml. This study demonstrated a direct relationship between predicted ATP-binding capacity of GspE and LT secretion, and between the latter and virulence.     

Occurrence of Genes Associated with Enterotoxigenic and Enterohemorrhagic Escherichia coli in Agricultural Waste Lagoons

The prevalence among all Escherichia coli bacteria of the LTIIa toxin gene and STII toxin gene, both associated with enterotoxigenic E. coli, and of three genes (stxI, stxII, and eaeA) associated with enterohemorrhagic E. coli was determined in farm waste disposal systems seasonally for 1 year. Single- and nested-PCR results for the number of E. coli isolates carrying each toxin gene trait were compared with a five-replicate most-probable-number (MPN) method. The STII and LTIIa toxin genes were present continuously at all farms and downstream waters that were tested. Nested-MPN-PCR manifested sensitivity increased over that of single-MPN-PCR by a factor of 32 for LTIIa, 10 for STII, and 2 for the stxI, stxII, and eaeA genes. The geometric mean prevalence of each toxin gene within the E. coli community in waste disposal site waters after nested MPN-PCR was 1:8.5 E. coli isolates (1:8.5 E. coli) for the LTIIa toxin gene and 1:4 E. coli for the STII toxin gene. The geometric mean prevalence for the simultaneous occurrence of toxin genes stxI, stxII, and eaeA, was 1:182 E. coli. These findings based on total population analysis suggest that prevalence rates for these genes are higher than previously reported in studies based on surveys of single isolates. With a population-based approach, the frequency of each toxin gene at the corresponding disposal sites and the endemic nature of diseases on farms can be easily assessed, allowing farmers and public health officials to evaluate the risk of infection to animals or humans.     

Phase 1 Evaluation of Intranasal Virosomal Influenza Vaccine with and without Escherichia coli Heat-Labile Toxin in Adult Volunteers

Virosomal vaccines were prepared by extracting hemagglutinin (HA) and neuraminidase from influenza virus and incorporating it in the membranes of liposomes composed of phosphatidylcholine. Two intranasal spray vaccine series were prepared: one series comprised 7.5 micrograms of HA of each of three strains recommended by the World Health Organization and 1 microgram of Escherichia coli heat-labile toxin (HLT), and the other contained the HA without HLT. In addition, a third vaccine preparation contained 15 micrograms of HA and 2 micrograms of HLT. The parenteral virosomal vaccine contained 15 micrograms of HA without additional adjuvant. The immunogenicity of a single spray vaccination (15 micrograms of HA and 2 micrograms of HLT) was compared with that of two vaccinations (7.5 micrograms of HA with or without 1 microgram of HLT) with an interval of 1 week in 60 healthy working adults. Twenty volunteers received one parenteral virosomal vaccine. Two nasal spray vaccinations with HLT-adjuvanted virosomal influenza vaccine induced a humoral immune response which was comparable to that with a single parenteral vaccination. A significantly higher induction of influenza virus-specific immunoglobulin A was noted in the saliva after two nasal applications. The immune response after a single spray vaccination was significantly lower. It could be shown that the use of HLT as a mucosal adjuvant is necessary to obtain a humoral immune response comparable to that with parenteral vaccination. All vaccines were well tolerated.     

Feed Fermentation with Reuteran- and Levan-Producing Lactobacillus reuteri Reduces Colonization of Weanling Pigs by Enterotoxigenic Escherichia coli

This study determined the effect of feed fermentation with Lactobacillus reuteri on growth performance and the abundance of enterotoxigenic Escherichia coli (ETEC) in weanling piglets. L. reuteri strains produce reuteran or levan, exopolysaccharides that inhibit ETEC adhesion to the mucosa, and feed fermentation was conducted under conditions supporting exopolysaccharide formation and under conditions not supporting exopolysaccharide formation. Diets were chosen to assess the impact of organic acids and the impact of viable L. reuteri bacteria. Fecal samples were taken throughout 3 weeks of feeding; at the end of the 21-day feeding period, animals were euthanized to sample the gut digesta. The feed intake was reduced in pigs fed diets containing exopolysaccharides; however, feed efficiencies did not differ among the diets. Quantification of L. reuteri by quantitative PCR (qPCR) detected the two strains used for feed fermentation throughout the intestinal tract. Quantification of E. coli and ETEC virulence factors by qPCR demonstrated that fermented diets containing reuteran significantly (P < 0.05) reduced the copy numbers of genes for E. coli and the heat-stable enterotoxin in feces compared to those achieved with the control diet. Any fermented feed significantly (P < 0.05) reduced the abundance of E. coli and the heat-stable enterotoxin in colonic digesta at 21 days; reuteran-containing diets reduced the copy numbers of the genes for E. coli and the heat-stable enterotoxin below the detection limit in samples from the ileum, the cecum, and the colon. In conclusion, feed fermentation with L. reuteri reduced the level of colonization of weaning piglets with ETEC, and feed fermentation supplied concentrations of reuteran that may specifically contribute to the effect on ETEC.     

Little Heterogeneity among Genes Encoding Heat-Labile and Heat-Stable Toxins of Enterotoxigenic Escherichia coli Strains Isolated from Diarrheal Pigs

To examine whether the heat-labile enterotoxin gene in porcine enterotoxigenic Escherichia coli (ETEC) strains is as divergent as in human ETEC strains, we sequenced the heat-labile and heat-stable toxin genes from 52 and 33 porcine ETEC strains, respectively. We found that the STa gene is identical, that the LT gene has only two mutations in 4 (of 52) strains, and that both mutations cause a reduction in GM1 binding and toxicity.Enterotoxigenic Escherichia coli (ETEC) strains that colonize small intestines and produce enterotoxins are the major cause of diarrheal disease in humans and animals (8, 16, 18, 21). The key virulence factors of ETEC in diarrhea include enterotoxins and colonization factors or adhesins. Colonization factors or adhesins mediate the attachment of bacteria to host epithelium cells and facilitate bacterial colonization. Enterotoxins disrupt fluid homeostasis and stimulate fluid hyper-secretion in the intestinal epithelial cells that results in diarrhea. Heat-labile toxin (LT) and heat-stable toxin (ST) are the main enterotoxins associated with diarrhea in humans and farm animals, but different LT and ST are produced by human and animal ETEC strains (9, 16).The LT produced by porcine ETEC strains (pLT) or human ETEC strains (hLT) is a holotoxin-structured protein that has one LT_A subunit and five LT_B subunits. Although pLT and hLT are highly homologous in structure and function, these two proteins differ antigenetically (9). Sequence comparative studies showed that the following seven amino acids are different between pLT and hLT: the 4th, 213th, and 237th amino acids of the A subunits and the 4th, 13th, 46th, and 102nd amino acids of the B subunits (6, 7). Similarly, STa (ST type 1) carried by human and porcine ETEC strains is also different. The STa associated with porcine diarrhea (pSTa) is a peptide of 18 amino acids, whereas the STa produced by human ETEC strains (hSTa) is 19 amino acids in length (5, 19). Despite the fact that ETEC constructs expressing pLT or hLT, and pSTa or hSTa, are equivalently virulent in causing diarrhea in gnotobiotic pigs (25), pLT and pSTa are typically expressed by porcine ETEC strains that only cause diarrhea in pigs, whereas hLT and hSTa are exclusively produced by human ETEC strains associated with diarrhea in humans. Although pLT and STb, another porcine-specific ST, were occasionally detected in ETEC strains isolated from human diarrheal patients (3), only infections with hSTa⁺, hLT⁺, or hSTa⁺/hLT⁺ ETEC strains cause diarrhea in humans (17).Interspecies LT have been intensively compared for molecular and immunological characteristics (4, 10, 20, 23). In contrast, intraspecies LT has not been studied much. For a long time, both pLT and hLT were assumed to be highly conserved. However, a very recent study showed that the hLT gene carried by human ETEC strains is considerably divergent (12). After restriction fragment length polymorphism analysis and DNA sequencing of 51 human ETEC strains, Lasaro et al. reported that the human LT gene had seven polymorphic restriction fragment length polymorphism types and 30 nucleotide polymorphic sites and recognized 16 different hLT types (12). To examine whether the LT gene carried by porcine ETEC strains has a similar heterogeneity, we PCR amplified and DNA sequenced the LT genes and also the STa genes of various ETEC strains isolated from diarrheal pigs and analyzed gene sequence conformity.Fifty-two porcine ETEC strains that express LT alone or LT together with other toxins (LT⁺/STb⁺, LT⁺/STb⁺/STa⁺, LT⁺/STb⁺/EAST1⁺, and LT⁺/STa⁺/STb⁺/EAST1⁺) and K88ac or F18 fimbria were selected for the sequencing of the LT gene. Those porcine ETEC strains were isolated from pigs with postweaning diarrhea at different farms in South Dakota, Iowa, Minnesota, Nebraska, and North Dakota. The eltAB gene encoding LT from these 52 strains was PCR amplified with primers pLT-F (5′-ATCCTCGCTAGCATGTTTTAT-3′) and pLT-R (5′-CCCCTCCGGCCGAGCTTAGTT-3′) (25). PCRs were performed in an MJ PT-100 thermocycler (Bio-Rad, Hercules, CA) in a reaction of 50 μl containing 1× Taq DNA polymerase buffer (with Mg²⁺), 0.2 mM deoxynucleoside triphosphate, 0.5 μM each forward and reverse primers, 100 ng of total genomic DNA, and 1 unit Taq DNA polymerase (Applied Biosystems, Foster City, CA). The PCR program contained one cycle of 2 min at 94°C; 30 cycles of 35 s at 94°C, 35 s at 52°C, and 2 min at 72°C; and an extension of 6 min at 72°C. The amplified PCR products were separated on 1% agarose gels (FMC Bioproducts, Rockland, MA) by electrophoresis and purified using a QIAquick gel extraction kit according to the manufacturer''s instructions (Qiagen, Valencia, CA). A mixture of purified PCR product (100 to 150 ng) and 10 pmol primer was sent to the Nevada Genomic Center at the University of Nevada for sequencing. Three primers, pLT-F, LT₁₉₂-F (5′-GATTCATCAAGAACAATCCACAGGTG-3′), and LT₁₉₂-R (5′-CCTGTGATTGTTCTTGATGAATC-3′), were used for sequencing the entire eltAB gene.The sequences of the eltAB gene from all 52 porcine ETEC strains were aligned and visually examined. We found that the eltAB gene was nearly identical among the sequenced porcine ETEC strains. Forty-eight (of 52) ETEC strains had identical gene sequences, and only four strains showed heterogeneity. The pathotypes of these four strains were K88/LT/STb, K88/LT/STb/STa, K88/LT/STb/EAST1, and F18/LT/STa/STb/Stx2e. Furthermore, only nucleotides coding two amino acids, the 44th (S44N) and the 60th (S60T) of the eltB gene encoding the B subunit, differed among these four strains. To our surprise, neither of these two substitutions were homologous to the hLT gene nor to any of the hLT types recognized by Lasaro et al. (12). Lasaro et al. showed that 11 of the 15 different hLT types shared some homology with pLT, and some hLT types had as many as four amino acids (K4R and K213E of LT_A and S4T, R13H, or A46E of LT_B; out of seven heterogeneous amino acids) homologous to pLT. Indeed, the hLT6 type differed from the LT of human ETEC prototype {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 in four amino acids (K4R and K213E of LT_A and S4T and A46E of LT_B) (12), but all four of these heterogeneous amino acids were homologous to pLT. Similarly, four of the five amino acids that differed from the prototype hLT in the hLT4 type were identical to pLT. That means that the hLT4 and hLT6 types had only three amino acids heterogeneous to pLT but four different residues compared to the hLT prototype. It seems that hLT4 and hLT6 are more likely pLT rather than hLT. Given that the divergence of the pLT and hLT genes is assumed to be a very recent evolutionary event that occurred 0.9 million years ago (23), it is likely that the hLT gene retains some pLT gene characters (amino acids) that could be of their common ancestor. However, a high homology in the pLT gene certainly seems unparallel to the evolution of the hLT gene. Our further sequence comparison indicated that S44N-substituted pLT_B [pLT_B(S44N)] is homologous to cholera toxin (CT). It has been suggested that the CT and LT genes were derived from the same ancestor but diverged to two lineages about 130 million years ago (23). Then, it is more likely that this pLT_B(S44N) represents a plesiomorphic character, meaning a primitive character that belongs to the common ancestor of CT and LT. The retention of this primitive pLT_B(S44N) by some porcine ETEC strains suggests that the pLT gene could have evolved at a relatively lower rate. Whether such a lower substitution rate of the LT gene in porcine ETEC strains is associated with a lower host exchange rate or a limited travel range in pigs is unclear to us. However, future studies to determine whether an increase in sampling sizes, by including porcine ETEC strains from a greater geographic coverage, could reveal a higher heterogeneity or a greater evolution rate in the pLT gene will be worthwhile.To examine whether the heterogeneity of pS44N and pS60T at the B subunit could affect the biological function of pLT, we cloned the native pLT gene into vector pBR322 (p8458), performed site-directed mutation of the eltAB gene for a substitution of S44N or S60T, and tested these two mutated LT proteins for their binding capability to GM1 receptors and their enterotoxic activity in stimulating intracellular cyclic AMP (cAMP) in cells. Primers pBRNheI-F2 (5′-CAGCATCGCCATTCACTATG-3′) and pBREagI-R (5′-AGATGACGACCATCAGGGAC-3′) were designed to amplify the porcine eltAB gene. The amplified eltAB gene products and vector pBR322 were digested with NheI and EagI (New England Biolabs, Beverly, MA), separated by gel electrophoresis, purified with the QIAquick gel extraction kit, and then ligated with T4 DNA ligase (Promega, Madison, WI). Two microliters of the T4-ligated products were introduced into 25 μl of TOPO cells (Invitrogen, Valencia, CA) in a standard electroporation. Antibiotic-selected colonies were initially screened by PCR, and positive colonies were sequenced to ensure that the cloned gene was in the reading frame. The verified clone was selected as a pLT recombinant strain and designated strain 8458. To construct mutant strains, two pairs of primers, LT_B44-F (5′-ATCATTACATTTAAGAACGGCGAA-3′) and LT_B44-R (5′-TTCGCCGTTCTTAAATGTAATGAT-3′) and LT_B60-F (5′-CAACATATAGACACCCAGAAAAAAGCC-3′) and LT_B60-R (5′-GGCTTTTTTCTGGGTGTCTATATGTTG-3′), were used for site-directed mutation at nucleotides coding the 44th and 60th amino acids of the LT_B subunit, respectively. Briefly, the amplified products from two separate PCRs, one using pBRNheI-F2 with LT_B44-R or LT_B60-R and the other using pBREagI-R with LT_B44-F or LT_B60-F, with recombinant pLT plasmid p8458 as the DNA template, were overlapped in a third splicing overlap extension PCR to produce mutated pLT genes. The splicing overlap extension PCR products were digested with NheI and EagI restriction enzymes and ligated into vector pBR322 for the p8647 (S44N) and p8649 (S60T) plasmids. Plasmids p8647 and p8649 were separately introduced into TOPO 10 E. coli cells (Invitrogen) for mutant strains 8647 (S44N) and 8649 (S60T).Equivalent amounts of cells from overnight-grown cultures of the recombinant (8458) and two mutant (8647 and 8649) strains were used for total protein preparation by using bacterial protein extraction reagent (B-PER in phosphate buffer; Pierce, Rockford, IL). Both pelleted protein samples (periplasmic proteins) and culture supernatant samples (outer-membrane secreted proteins) were used in a GM1 enzyme-linked immunosorbent assay (ELISA) to examine whether a substitution at the 44th or 60th amino acid would affect the binding of LT to GM1 receptors. Anti-CT rabbit antiserum (1:5,000; Sigma) and horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (1:5,000; Sigma, St. Louis, MO) were used as the primary and secondary antibodies as described previously (2, 14, 24). GM1 ELISA data indicated optical density (OD) values from the pellet samples of strains 8548, 8647, and 8649 and phosphate-buffered saline of 0.677 ± 0.004, 0.616 ± 0.001, 0.647 ± 0.004, and 0.006 ± 0, whereas the OD values of the supernatant samples which were vacuum concentrated were 0.949 ± 0.008, 0.726 ± 0.004, 0.660 ± 0.005, and 0.05 ± 0.002, respectively (Fig. ​(Fig.1).1). Statistical analysis using the Student t test with two-tailed distribution indicated that the binding of the pellet samples from the native and the mutated LT to GM1 was not significantly different (P = 0.10 and P = 0.45, respectively). However, the GM1 binding from the supernatant samples of the LT mutant strains was significantly lower than that of the LT recombinant strain (P < 0.01 and P < 0.01, respectively).Open in a separate windowFIG. 1.GM1 ELISA to detect LT proteins expressed by the pLT recombinant (8458) and mutant [8647(S44N) and 8649(S60T)] strains. Protein samples from the pellet and vacuum-concentrated supernatants of overnight-grown cultures were used in the GM1 ELISA. Each sample was assayed in triplicate to calculate OD means and standard deviations. Anti-CT serum (1:5,000) was used as the primary antibody and goat anti-rabbit horseradish peroxidase-conjugated immunoglobulin G (1:5,000) was used as the secondary antibody. OD values were measured after a 20-min reaction with peroxidase substrates (KPL, Gaithersburg, MD) at a wavelength of 405 nm.Our GM1 ELISA data indicated that the supernatant sample of the recombinant strain expressing a native LT had a greater GM1 binding activity. This could suggest that the recombinant strain had more LT protein crossing the outer membrane and being secreted in the supernatant than either mutant strain or that mutations at the B subunit negatively affected the binding of LT proteins to GM1 receptors. It has been reported that a single amino acid mutation of the LT_B or CT_B subunit resulted in lower GM1 binding activity, especially mutations of residues from the binding pocket (13, 15, 22). When amino acid 33 or 88 of the CT_B subunit was replaced, both mutants failed to bind or bound poorly to GM1 (22), and when a substitution at residue 57 of its B subunit occurred, this CT mutant showed 1.5-log-lower GM1 binding than the native CT (1, 13). Similarly, when amino acid 46 or 47 of the B subunit was replaced, both LT mutants exhibited lower GM1 binding activity than the wild-type LT strain (13). However, in contrast to our observation that our 8647 and 8649 mutant strains showed lower GM1 binding activity in the supernatant, Mudrak et al. indicated that the T47A mutant strain had more LT protein detected in the supernatant than the wild-type strain (13). Whether and how a mutation at amino acid 44 or 60 of the B subunit affects the formation, stability, or secretion of the mutant LT proteins will be studied in the future.To examine whether the lower GM1 binding activity of the supernatant samples from the mutant strains was caused by a lower LT production, we conducted an ELISA by directly coating an ELISA plate with total proteins from the pellet and supernatant samples of each strain (without GM1) and by using anti-CT antiserum to quantify the LT protein. ELISA data showed that the OD values of strains 8458, 8647, and 8649 were 0.209 ± 0.005, 0.225 ± 0.009, and 0.21 ± 0 in the supernatant samples and 0.571 ± 0.025, 0.614 ± 0.060, and 0.616 ± 0.026 in the pellet samples, respectively. A Student t test indicated that there were no significant differences between the recombinant strain and the mutant strains in the OD values for the pellet and supernatant protein samples (P = 0.26 and P = 0.84, respectively, for the supernatant samples; P = 0.34 and P = 0.10, respectively, for the pellet samples). These data suggested that a similar amount of LT proteins was produced among these three strains.A single amino acid substitution of the B subunit can result in a reduction in not only GM1 binding but also toxicity for the mutated LT proteins (11, 13, 22). To study whether the mutation of S44N or S60T at the B subunit affected pLT toxicity, we measured the recombinant and mutant strains for their stimulation of intracellular cAMP levels in T-84 cells by using a cAMP competitive enzyme immunoassay (EIA) kit (Invitrogen) by following the manufacturer''s instructions. Briefly, 1 × 10⁵ T-84 cells were seeded in each well of a 24-well plate. After removing the Dulbecco''s modified Eagle medium (DMEM/F12; Gibco/Invitrogen, Grand Island, NY), 75 μl of overnight-grown (in 4AA medium) supernatant of the recombinant or each mutant strain (in triplicate) was added to each well. The cells were lysed with 100 μl of 0.1 M HCl after 2 h of incubation and then neutralized. A total of 100 μl of lysis supernatant was mixed with kit-supplied conjugates and antibody reagents, and the mixture was added to each well of the supplied EIA plate. After incubation on a shaker at 500 rpm at room temperature for 2 h, the plate was washed and dried by blotting, and p-nitrophenyl phosphate substrate solution was added. The OD was measured at 405 nm after 20 min of development. Data from the cAMP ELISA indicated that cAMP levels in T-84 cells incubated with supernatant samples from strains 8458, 8647, and 8649 (from equivalent amounts of cells) were 2.3 ± 0.1, 0.46 ± 0.05, and 0.35 ± 0.01 pmol/ml, respectively (Fig. ​(Fig.2).2). Data clearly indicated that the mutations of S44N and S60T reduced the LT toxic activity. Knowing that it is the A subunit that determines the toxicity of LT and CT, whereas the LT_B and CT_B subunits mediate the binding of the toxin to the host GM1 receptors, we thought that substitution at the B subunits would not affect toxicity. However, we believe that mutations at the B subunits could alter LT protein structure and reduce the binding of the holotoxin to the host GM1 receptors, thus resulting in the reduction of toxic activity.Open in a separate windowFIG. 2.Intracellular cAMP ELISA to detect the toxicity of native LT and mutated LT proteins. Supernatants (in 4AA medium) of overnight-grown cultures from the 8458 (recombinant), 8647 (S44N), and 8649 (S60T) strains were used to stimulate an increase in intracellular cAMP levels in T-84 cells by using a cyclic GMP EIA kit (Invitrogen).The estA gene encoding STa from 33 STa-positive porcine ETEC strains was also sequenced for conformity. This porcine estA gene was PCR amplified using primers pSTaSfcI-F2 and STaEagI-R under conditions described previously (25). The PCR products were purified and sequenced with pSTaSfcI-F2 primer. The sequencing data showed that all sampled STa genes were identical and of porcine origin.Sequence data from our study clearly indicated that both LT and STa expressed by porcine ETEC strains are porcine specific. The LT gene of porcine ETEC strains showed little heterogeneity, and the STa gene is identical. Information from this study will be helpful for a prevalence study of toxin genes among porcine ETEC strains and toxin gene evolution and possibly instructive in antitoxin vaccine development. However, future studies with increasing sampling sizes and a greater geographic coverage will be helpful to understand divergence in the LT and STa genes among porcine ETEC strains.     

Glucose Significantly Enhances Enterotoxigenic Escherichia coli Adherence to Intestinal Epithelial Cells through Its Effects on Heat-Labile Enterotoxin Production

The present study tested whether exposure of enterotoxigenic Escherichia coli (ETEC) to glucose at different concentrations in the media results in increased bacterial adherence to host cells through increased heat-labile enterotoxin (LT) production, thereby suggesting the effects are physiological. Porcine-origin ETEC strains grown in Casamino acid yeast extract medium containing different concentrations of glucose were washed and inoculated onto IPEC-J2 porcine intestinal epithelial cells to test for effects on adherence and host cell cAMP concentrations. Consistent with previous studies, all LT⁺ strains had higher ETEC adherence to IPEC-J2 cells than did LT⁻ strains. Adherence of the LT⁻ but not the LT⁺ strains was increased by pre-incubating the IPEC-J2 cells with LT and decreased by co-incubation with GM1 ganglioside in a dose-dependent manner (P<0.05). To determine whether the glucose concentration of the cell culture media has an effect on adherence, IPEC-J2 cells were inoculated with LT⁺ or LT⁻ strains in cell culture media containing a final glucose concentration of 0, 0.25, 0.5, 1.0 or 2.0%, and incubated for 4 h. Only media containing 0.25% glucose resulted in increased adherence and cAMP levels, and this was limited to IPEC-J2 cells inoculated with LT⁺ strains. This study supports the hypothesis that glucose, at a concentration optimal for LT expression, enhances bacterial adherence through the promotion of LT production. Hence, these results establish the physiological relevance of the effects of glucose on LT production and provide a basis for how glucose intake may influence the severity of ETEC infection.     

Genetic Rearrangements of the Regions Adjacent to Genes Encoding Heat-Labile Enterotoxins (eltAB) of Enterotoxigenic Escherichia coli Strains

One of the most common bacterially mediated diarrheal infections is caused by enterotoxigenic Escherichia coli (ETEC) strains. ETEC-derived plasmids are responsible for the distribution of the genes encoding the main toxins, namely, the heat-labile and heat-stable enterotoxins. The origins and transfer modes (intra- or interplasmid) of the toxin-encoding genes have not been characterized in detail. In this study, we investigated the DNA regions located near the heat-labile enterotoxin-encoding genes (eltAB) of several clinical isolates. It was found that the eltAB region is flanked by conserved 236- and 280-bp regions, followed by highly variable DNA sequences which consist mainly of partial insertion sequence (IS) elements. Furthermore, we demonstrated that rearrangements of the eltAB region of one particular isolate, which harbors an IS91R sequence next to eltAB, could be produced by a recA-independent but IS91 sequence-dependent mechanism. Possible mechanisms of dissemination of IS element-associated enterotoxin-encoding genes are discussed.     

Effect of Lactobacillus rhamnosus NCDC 298 with FOS in Combination on Viability and Toxin Production of Enterotoxigenic Escherichia coli

Probiotics and Antimicrobial Proteins - The present study was to investigate the utilization of prebiotics by Lactobacillus rhamnosus NCDC 298 and its synergistic adversary effect on both...     

LeoA,B and C from Enterotoxigenic Escherichia coli (ETEC) Are Bacterial Dynamins

Escherichia coli (ETEC) strain {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 contains a GTPase virulence factor, LeoA, which is encoded on a pathogenicity island and has been shown to enhance toxin release, potentially through vesicle secretion. By sequence comparisons and X-ray structure determination we now identify LeoA as a bacterial dynamin-like protein (DLP). Proteins of the dynamin family remodel membranes and were once thought to be restricted to eukaryotes. In ETEC {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 LeoA localises to the periplasm where it forms a punctate localisation pattern. Bioinformatic analyses of leoA and the two upstream genes leoB and leoC suggest that LeoA works in concert with a second dynamin-like protein, made up of LeoB and LeoC. Disruption of the leoAB genes leads to a reduction in secretion of periplasmic Tat-GFP and outer membrane OmpA. Our data suggest a role for LeoABC dynamin-like proteins in potentiating virulence through membrane vesicle associated toxin secretion.     

肠毒素大肠杆菌定居因子抗原基因CFA-I在痢疾杆菌中的表达

通过体外重组的方法 ,构建了包含asd基因的重组表达质粒 pZHY2 1,与福氏志贺氏菌Fwl0 1构成了宿主 载体平衡致死系统 ,Western印迹结果表明 ,在没有抗生素条件选择的情况下 ,稳定表达肠毒素大肠杆菌定居因子抗原CFA I。电镜结果显示 ,在重组菌株的菌体表面 ,表达产物能够装配成菌毛。重组菌通过口服和鼻饲免疫小鼠后 ,可以诱生CFA I的抗体 ;同时可以检测到分泌型IgA产生 ,表明重组菌可以诱导相应的粘膜免疫反应     

Toxin-Antitoxin Systems Are Important for Niche-Specific Colonization and Stress Resistance of Uropathogenic Escherichia coli

Toxin-antitoxin (TA) systems are prevalent in many bacterial genomes and have been implicated in biofilm and persister cell formation, but the contribution of individual chromosomally encoded TA systems during bacterial pathogenesis is not well understood. Of the known TA systems encoded by Escherichia coli, only a subset is associated with strains of extraintestinal pathogenic E. coli (ExPEC). These pathogens colonize diverse niches and are a major cause of sepsis, meningitis, and urinary tract infections. Using a murine infection model, we show that two TA systems (YefM-YoeB and YbaJ-Hha) independently promote colonization of the bladder by the reference uropathogenic ExPEC isolate CFT073, while a third TA system comprised of the toxin PasT and the antitoxin PasI is critical to ExPEC survival within the kidneys. The PasTI TA system also enhances ExPEC persister cell formation in the presence of antibiotics and markedly increases pathogen resistance to nutrient limitation as well as oxidative and nitrosative stresses. On its own, low-level expression of PasT protects ExPEC from these stresses, whereas overexpression of PasT is toxic and causes bacterial stasis. PasT-induced stasis can be rescued by overexpression of PasI, indicating that PasTI is a bona fide TA system. By mutagenesis, we find that the stress resistance and toxic effects of PasT can be uncoupled and mapped to distinct domains. Toxicity was specifically linked to sequences within the N-terminus of PasT, a region that also promotes the development of persister cells. These results indicate discrete, multipurpose functions for a TA-associated toxin and demonstrate that individual TA systems can provide bacteria with pronounced fitness advantages dependent on toxin expression levels and the specific environmental niche occupied.     

大肠杆菌耐热性肠毒素STI三重串联突变体与黏附素K99融合基因的表达研究

产肠毒素大肠杆菌（ETEC）是一种导致仔畜和婴儿腹泻的主要病原之一,它的毒力因子主要有两类：黏附素（CFAs）和耐热性肠毒素（ST）或不耐热性肠毒素（LT）。通过PCR技术及双酶切连接技术,成功构建了含有3个STI突变体和1个黏附素K99基因的重组表达质粒pE3S(S)LK和pE3S(G)LK。重组菌株BL21(DE3) (pE3S(S)LK)和BL21(DE3)(pE3S(G)LK)的表达产物经SDS-PAGE和免疫印迹分析,表明以上两种重组菌株均能高效表达3STI(S)-K99和3STI(G)-K99融合蛋白,且融合蛋白能够被产肠毒素性大肠杆菌强毒株C83922 抗血清特异性识别。其次,利用乳鼠灌胃实验检测重组蛋白的生物学毒性,结果均为阴性(G/C值≤0.083),这表明该菌株已无STI生物学毒性。这些为研发预防大肠杆菌性腹泻的新型高效多价基因工程疫苗提供了基本素材和理论指导。     

Rotavirus 2/6 Viruslike Particles Administered Intranasally with Cholera Toxin, Escherichia coli Heat-Labile Toxin (LT), and LT-R192G Induce Protection from Rotavirus Challenge

We have shown that rotavirus 2/6 viruslike particles composed of proteins VP2 and VP6 (2/6-VLPs) administered to mice intranasally with cholera toxin (CT) induced protection from rotavirus challenge, as measured by virus shedding. Since it is unclear if CT will be approved for human use, we evaluated the adjuvanticity of Escherichia coli heat-labile toxin (LT) and LT-R192G. Mice were inoculated intranasally with 10 μg of 2/6-VLPs combined with CT, LT, or LT-R192G. All three adjuvants induced equivalent geometric mean titers of rotavirus-specific serum antibody and intestinal immunoglobulin G (IgG). Mice inoculated with 2/6-VLPs with LT produced significantly higher titers of intestinal IgA than mice given CT as the adjuvant. All mice inoculated with 2/6-VLPs mixed with LT and LT-R192G were totally protected (100%) from rotavirus challenge, while mice inoculated with 2/6-VLPs mixed with CT showed a mean 91% protection from challenge. The availability of a safe, effective mucosal adjuvant such as LT-R192G will increase the practicality of administering recombinant vaccines mucosally.     

Escherichia coli O157:H7 and Other E. coli Strains Share Physiological Properties Associated with Intestinal Colonization

Escherichia coli isolates (72 commensal and 10 O157:H7 isolates) were compared with regard to physiological and growth parameters related to their ability to survive and persist in the gastrointestinal tract and found to be similar. We propose that nonhuman hosts in E. coli O157:H7 strains function similarly to other E. coli strains in regard to attributes relevant to gastrointestinal colonization.Escherichia coli is well known for its ecological versatility (15). A life cycle which includes both gastrointestinal and environmental stages has been stressed by both Savageau (15) and Adamowicz et al. (1). The gastrointestinal stage would be subjected to acid and detergent stress. The environmental stage is implicit in E. coli having transport systems for fungal siderophores (4) as well as pyrroloquinoline quinone-dependent periplasmic glucose utilization (1) because their presence indicates evolution in a location containing fungal siderophores and pyrroloquinoline quinone (1).Since its recognition as a food-borne pathogen, there have been numerous outbreaks of food-borne infection due to E. coli O157:H7, in both ground beef and vegetable crops (6, 13). Cattle are widely considered to be the primary reservoir of E. coli O157:H7 (14), but E. coli O157:H7 does not appear to cause disease in cattle. To what extent is E. coli O157:H7 physiologically unique compared to the other naturally occurring E. coli strains? We feel that the uniqueness of E. coli O157:H7 should be evaluated against a backdrop of other wild-type E. coli strains, and in this regard, we chose the 72-strain ECOR reference collection originally described by Ochman and Selander (10). These strains were chosen from a collection of 2,600 E. coli isolates to provide diversity with regard to host species, geographical distribution, and electromorph profiles at 11 enzyme loci (10).In our study we compared the 72 strains of the ECOR collection against 10 strains of E. coli O157:H7 and six strains of E. coli which had been in laboratory use for many years (Table ​(Table1).1). The in vitro comparisons were made with regard to factors potentially relevant to the bacteria''s ability to colonize animal guts, i.e., acid tolerance, detergent tolerance, and the presence of the Entner-Doudoroff (ED) pathway (Table ​(Table2).2). Our longstanding interest in the ED pathway (11) derives in part from work by Paul Cohen''s group (16, 17) showing that the ED pathway is important for E. coli colonization of the mouse large intestine. Growth was assessed by replica plating 88 strains of E. coli under 40 conditions (Table ​(Table2).2). These included two LB controls (aerobic and anaerobic), 14 for detergent stress (sodium dodecyl sulfate [SDS], hexadecyltrimethylammonium bromide [CTAB], and benzalkonium chloride, both aerobic and anaerobic), 16 for acid stress (pH 6.5, 6.0, 5.0, 4.6, 4.3, 4.2, 4.1, and 4.0), four for the ability to grow in a defined minimal medium (M63 glucose salts with and without thiamine), and four for the presence or absence of a functional ED pathway (M63 with gluconate or glucuronate). All tests were done with duplicate plates in two or three separate trials. The data are available in Tables S1 to S14 in the supplemental material, and they are summarized in Table ​Table22.

TABLE 1.

E. coli strains used in this study

Comparison between Enterotoxigenic Escherichia coli Strains Expressing “F42,” F41 and K99 Colonization Factors

Two enterotoxigenic Escherichia coli (ETEC) strains (coded 567/7 and 103) isolated from piglets with neonatal diarrhea were described as producers of a new adhesin (F42). With the use of molecular biology and immunology techniques such as DNA hybridization with probes for F41 and K99 genes and Western-blotting of the superficial proteins of these strains and standard E. coli strains carrying genes for F41 and K99 adhesins, it was demonstrated that this new adhesin either shares extensive genetic and immunological determinants with F41 adhesin or they are the same fimbriae.