Dose-Dependent Effects of Lactobacillus rhamnosus on Serum Interleukin-17 Production and Intestinal T-Cell Responses in Pigs Challenged with Escherichia coli

The mechanism underlying the dose effect of probiotics on ameliorating diarrhea has not been fully elucidated. Here, low (1 × 10⁹ CFU/ml) or high (1 × 10¹¹ CFU/ml) doses of Lactobacillus rhamnosus ATCC 7469 were administered orally to piglets for 1 week before F4 (K88)-positive enterotoxigenic Escherichia coli (F4⁺ ETEC) challenge. Administration of a low, but not a high, dose of L. rhamnosus decreased the percentage of CD3⁺ CD4⁺ CD8⁻ T cells in the peripheral blood. Notably, transiently increased serum concentrations of interleukin-17A (IL-17A) were observed after F4⁺ ETEC challenge in pigs pretreated with a high dose of L. rhamnosus. Administration of L. rhamnosus increased the percentage of the small intestinal lamina propria CD3⁺ CD4⁺ CD8⁻ cells and Peyer''s patch CD3⁺ CD4⁻ CD8⁻ and CD3⁻ CD4⁻ CD8⁺ cells. The percentage of ileal intraepithelial CD3⁺ CD4⁻ CD8⁺ cells increased only in the high-dose piglets. Administration of L. rhamnosus downregulated expression of ileal IL-17A after F4⁺ ETEC challenge but had no effect on expression of gamma interferon (IFN-γ), IL-12, IL-4, and FOXP3 mRNA in the small intestine. Expression of jejunal IL-2, ileal transforming growth factor β1 (TGF-β1), and ileal IL-10 was upregulated in the low-dose piglets after F4⁺ ETEC challenge. Our findings suggest that amelioration of infectious diarrhea in piglets by L. rhamnosus is associated with the generation of lamina propria CD3⁺ CD4⁺ CD8⁻ T cells, the expansion of Peyer''s patch CD3⁺ CD4⁻ CD8⁻ and CD3⁻ CD4⁻ CD8⁺ cells, and the attenuation of F4⁺ ETEC-induced increase in CD3⁺ CD4⁺ CD8⁺ T cells in the small intestine. However, consumption of high doses of L. rhamnosus may increase levels of serum IL-17A after F4⁺ ETEC challenge, thus eliciting a strong proinflammatory response.     

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.     

A Selected Lactobacillus rhamnosus Strain Promotes EGFR-Independent Akt Activation in an Enterotoxigenic Escherichia coli K88-Infected IPEC-J2 Cell Model

Enterotoxigenic Escherichia coli (ETEC) are important intestinal pathogens that cause diarrhea in humans and animals. Although probiotic bacteria may protect against ETEC-induced enteric infections, the underlying mechanisms are unknown. In this study, porcine intestinal epithelial J2 cells (IPEC-J2) were pre-incubated with and without Lactobacillus rhamnosus ATCC 7469 and then exposed to F4⁺ ETEC. Increases in TLR4 and NOD2 mRNA expression were observed at 3 h after F4⁺ ETEC challenge, but these increases were attenuated by L. rhamnosus treatment. Expression of TLR2 and NOD1 mRNA was up-regulated in cells pre-treated with L. rhamnosus. Pre-treatment with L. rhamnosus counteracted F4⁺ ETEC-induced increases in TNF-α concentration. Increased PGE₂. concentrations were observed in cells infected with F4⁺ ETEC and in cells treated with L. rhamnosus only. A decrease in phosphorylated epidermal growth factor receptor (EGFR) was observed at 3 h after F4⁺ ETEC challenge in cells treated with L. rhamnosus. Pre-treatment with L. rhamnosus enhanced Akt phosphorylation and increased ZO-1 and occludin protein expression. Our findings suggest that L. rhamnosus protects intestinal epithelial cells from F4⁺ ETEC-induced damage, partly through the anti-inflammatory response involving synergism between TLR2 and NOD1. In addition, L. rhamnosus promotes EGFR-independent Akt activation, which may activate intestinal epithelial cells in response to bacterial infection, in turn increasing tight junction integrity and thus enhancing the barrier function and restricting pathogen invasion. Pre-incubation with L. rhamnosus was superior to co-incubation in reducing the adhesion of F4⁺ ETEC to IPEC-J2 cells and subsequently attenuating F4⁺ ETEC-induced mucin layer destruction and suppressing apoptosis. Our data indicate that a selected L. rhamnosus strain interacts with porcine intestinal epithelial cells to maintain the epithelial barrier and promote intestinal epithelial cell activation in response to bacterial infection, thus protecting cells from the deleterious effects of F4⁺ ETEC.     

Bacterial Cell Division Regulation: Physiological Effects of Crystal Violet on Escherichia coli lon+ and lon− Strains

The Escherichia coli lon⁻ mutants apparently are defective in the ability to recommence cell division after temporary periods of deoxyribonucleic acid (DNA) synthesis inhibition. They are also more susceptible to cell division inhibition by the basic dye, crystal violet (CV), than are lon⁺ strains. In enriched broth, the lon⁺ strain continued to grow and divide in the presence of CV, but lon⁻ cell division was inhibited and filamentous growth resulted. In a supplemented minimal medium containing CV, lon⁻ cell division was only temporarily inhibited. There was no detectable specific effect on DNA synthesis, although CV slowed the rate of mass increase in both media. Trichloroacetic acid-insoluble lipid synthesis was preferentially inhibited in both lon⁺ and lon⁻ strains. In CV-containing enriched broth, diaminopimelic acid incorporation into trichloroacetic acid-insoluble compounds occurred at a rate greater than the rate of mass increase in both lon⁺ and lon⁻ strains. In a CV-containing supplemented minimal medium, diaminopimelic acid was incorporated to a greater extent by lon⁻ cells than by lon⁺ cells.     

Heat-labile enterotoxin promotes Escherichia coli adherence to intestinal epithelial cells

Given recent evidence suggesting that the heat-labile enterotoxin (LT) provides a colonization advantage for enterotoxigenic Escherichia coli (ETEC) in vivo, we hypothesized that LT preconditions the host intestinal epithelium for ETEC adherence. To test this hypothesis, we used an in vitro model of ETEC adherence to examine the role of LT in promoting bacterium-host interactions. We present data demonstrating that elaboration of LT promotes a significant increase in E. coli adherence. This phenotype is primarily dependent on the inherent ADP-ribosylation activity of this toxin, with a secondary role observed for the receptor-binding LT-B subunit. Rp-3′,5′-cyclic AMP (cAMP), an inhibitor of protein kinase A, was sufficient to abrogate LT's ability to promote subsequent bacterial adherence. Increased adherence was not due to changes in the surface expression of the host receptor for the K88ac adhesin. Evidence is also presented for a role for bacterial sensing of host-derived cAMP in promoting adherence to host cells.     

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.     

Dissimilatory Reduction of NO2− to NH4+ and N2O by a Soil Citrobacter sp.

Dissimilatory reduction of NO₂⁻ to N₂O and NH₄⁺ by a soil Citrobacter sp. was studied in an attempt to elucidate the physiological and ecological significance of N₂O production by this mechanism. In batch cultures with defined media, NO₂⁻ reduction to NH₄⁺ was favored by high glucose and low NO₃⁻ concentrations. Nitrous oxide production was greatest at high glucose and intermediate NO₃⁻ concentrations. With succinate as the energy source, little or no NO₂⁻ was reduced to NH₄⁺ but N₂O was produced. Resting cell suspensions reduced NO₂⁻ simultaneously to N₂O and free extracellular NH₄⁺. Chloramphenicol prevented the induction of N₂O-producing activity. The K_m for NO₂⁻ reduction to N₂O was estimated to be 0.9 mM NO₂⁻, yet the apparent K_m for overall NO₂⁻ reduction was considerably lower, no greater than 0.04 mM NO₂⁻. Activities for N₂O and NH₄⁺ production increased markedly after depletion of NO₃⁻ from the media. Amendment with NO₃⁻ inhibited N₂O and NH₄⁺ production by molybdate-grown cells but not by tungstate-grown cells. Sulfite inhibited production of NH₄⁺ but not of N₂O. In a related experiment, three Escherichia coli mutants lacking NADH-dependent nitrite reductase produced N₂O at rates equal to the wild type. These observations suggest that N₂O is produced enzymatically but not by the same enzyme system responsible for dissimilatory reduction of NO₂⁻ to NH₄⁺.     

Fusarium Wilt Suppression and Agglutinability of Pseudomonas putida

Mutants of Pseudomonas putida (Agg⁻) that lack the ability to agglutinate with components present in washes of bean and cucumber roots showed limited potential to protect cucumber plants against Fusarium oxysporum f. sp. cucumerinum. However, a higher level of protection was observed against Fusarium wilt in cucumber plants coinoculated with the parental bacterium (Agg⁺), which was agglutinable. The Agg⁻ mutants did not colonize the roots of cucumber plants as extensively as the Agg⁺ parental isolate did. In competition experiments involving bean roots inoculated with a mixture of Agg⁺ and Agg⁻ bacteria, the Agg⁺ strains colonized roots to a greater extent than the Agg⁻ cells did. These data suggest that the Agg⁺ phenotype provides additional interactions that aid in the beneficial character of P. putida.     

Polyphosphate Degradation in Stationary Phase Triggers Biofilm Formation via LuxS Quorum Sensing System in Escherichia coli

In most natural environments, association with a surface in a structure known as biofilm is the prevailing microbial life-style of bacteria. Polyphosphate (polyP), an ubiquitous linear polymer of hundreds of orthophosphate residues, has a crucial role in stress responses, stationary-phase survival, and it was associated to bacterial biofilm formation and production of virulence factors. In previous work, we have shown that Escherichia coli cells grown in media containing a critical phosphate concentration >37 mM maintained an unusual high polyP level in stationary phase. The aim of the present work was to analyze if fluctuations in polyP levels in stationary phase affect biofilm formation capacity in E. coli. Polymer levels were modulated by the media phosphate concentration or using mutant strains in polyP metabolism. Cells grown in media containing phosphate concentrations higher than 25 mM were defective in biofilm formation. Besides, there was a disassembly of 24 h preformed biofilm by the addition of high phosphate concentration to the medium. These phenotypes were related to the maintenance or re-synthesis of polyP in stationary phase in static conditions. No biofilm formation was observed in ppk⁻ppx⁻ or ppk⁻ppx⁻/ppk⁺ strains, deficient in polyP synthesis and hydrolysis, respectively. luxS and lsrK mutants, impaired in autoinducer-2 quorum sensing signal metabolism, were unable to form biofilm unless conditioned media from stationary phase wild type cells grown in low phosphate were used. We conclude that polyP degradation is required for biofilm formation in sufficient phosphate media, activating or triggering the production of autoinducer-2. According to our results, phosphate concentration of the culture media should be carefully considered in bacterial adhesion and virulence studies.     

Escherichia coli Expressing EAST1 Toxin Did Not Cause an Increase of cAMP or cGMP Levels in Cells, and No Diarrhea in 5-Day Old Gnotobiotic Pigs

Background

Enterotoxigenic Escherichia coli (ETEC) strains are the leading bacterial cause of diarrhea to humans and farm animals. These ETEC strains produce heat-labile toxin (LT) and/or heat-stable toxins that include type I (STa), type II (STb), and enteroaggregative heat-stable toxin 1 (EAST1). LT, STa, and STb (in pigs) are proven the virulence determinants in ETEC diarrhea. However, significance of EAST1 in ETEC-associated diarrheal has not been determined, even though EAST1 is highly prevalent among ETEC strains.

Methodology/Principal Findings

In this study, we constructed E. coli strains to express EAST1 toxin as the only toxin and studied them in cell lines and five-day old gnotobiotic piglets to determine significance of EAST1 toxin. Data from in vitro studies indicated that EAST1 did not stimulate an increase of intracellular cyclic AMP or GMP levels in T-84 cells or porcine cell line IPEC-J2, nor did it enhance LT or STa toxin of ETEC strains in stimulation of cAMP or cGMP in T-84 cells. In addition, 5-day old gnotobiotic pigs challenged with E. coli strains expressing EAST1 as the only toxin did not developed diarrhea or signs of clinical disease during 72 h post-inoculation.

Conclusion/Significance

Results from this study indicated that EAST1 alone is not sufficient to cause diarrhea in five-day old gnotobiotic pigs, and suggest that EAST1 likely is not a virulence determinant in ETEC-associated diarrhea.     

Allele Variants of Enterotoxigenic Escherichia coli Heat-Labile Toxin Are Globally Transmitted and Associated with Colonization Factors

Enterotoxigenic Escherichia coli (ETEC) is a significant cause of morbidity and mortality in the developing world. ETEC-mediated diarrhea is orchestrated by heat-labile toxin (LT) and heat-stable toxins (STp and STh), acting in concert with a repertoire of more than 25 colonization factors (CFs). LT, the major virulence factor, induces fluid secretion after delivery of a monomeric ADP-ribosylase (LTA) and its pentameric carrier B subunit (LTB). A study of ETEC isolates from humans in Brazil reported the existence of natural LT variants. In the present study, analysis of predicted amino acid sequences showed that the LT amino acid polymorphisms are associated with a geographically and temporally diverse set of 192 clinical ETEC strains and identified 12 novel LT variants. Twenty distinct LT amino acid variants were observed in the globally distributed strains, and phylogenetic analysis showed these to be associated with different CF profiles. Notably, the most prevalent LT1 allele variants were correlated with major ETEC lineages expressing CS1 + CS3 or CS2 + CS3, and the most prevalent LT2 allele variants were correlated with major ETEC lineages expressing CS5 + CS6 or CFA/I. LTB allele variants generally exhibited more-stringent amino acid sequence conservation (2 substitutions identified) than LTA allele variants (22 substitutions identified). The functional impact of LT1 and LT2 polymorphisms on virulence was investigated by measuring total-toxin production, secretion, and stability using GM1–enzyme-linked immunosorbent assays (GM1-ELISA) and in silico protein modeling. Our data show that LT2 strains produce 5-fold more toxin than LT1 strains (P < 0.001), which may suggest greater virulence potential for this genetic variant. Our data suggest that functionally distinct LT-CF variants with increased fitness have persisted during the evolution of ETEC and have spread globally.     

TNF-α and CD8+ T Cells Mediate the Beneficial Effects of Nitric Oxide Synthase-2 Deficiency in Pulmonary Paracoccidioidomycosis

Background

Nitric oxide (NO), a key antimicrobial molecule, was previously shown to exert a dual role in paracoccidioidomycosis, an endemic fungal infection in Latin America. In the intravenous and peritoneal models of infection, NO production was associated with efficient fungal clearance but also with non-organized granulomatous lesions. Because paracoccidioidomycosis is a pulmonary infection, we aimed to characterize the role of NO in a pulmonary model of infection.

Methodology/Principal Findings

C57Bl/6 wild type (WT) and iNOS^−/− mice were i.t. infected with 1×10⁶ Paracoccidioides brasiliensis yeasts and studied at several post-infection periods. Unexpectedly, at week 2 of infection, iNOS^−/− mice showed decreased pulmonary fungal burdens associated with an M2-like macrophage profile, which expressed high levels of TGF-β impaired ability of ingesting fungal cells. This early decreased fungal loads were concomitant with increased DTH reactions, enhanced TNF-α synthesis and intense migration of activated macrophages, CD4⁺ and CD8⁺ T cells into the lungs. By week 10, iNOS^−/− mice showed increased fungal burdens circumscribed, however, by compact granulomas containing elevated numbers of activated CD4⁺ T cells. Importantly, the enhanced immunological reactivity of iNOS^−/− mice resulted in decreased mortality rates. In both mouse strains, depletion of TNF-α led to non-organized lesions and excessive influx of inflammatory cells into the lungs, but only the iNOS^−/− mice showed increased mortality rates. In addition, depletion of CD8⁺ cells abolished the increased migration of inflammatory cells and decreased the number of TNF-α and IFN-γ CD4⁺ and CD8⁺ T cells into the lungs of iNOS^−/− mice.

Conclusions/Significance

Our study demonstrated that NO plays a deleterious role in pulmonary paracoccidioidomycosis due to its suppressive action on TNF-α production, T cell immunity and organization of lesions resulting in precocious mortality of mice. It was also revealed that uncontrolled fungal growth can be overcome by an efficient immune response.     

Type I fimbriae mediate in vitro adherence of porcine F18ac+ enterotoxigenic <Emphasis Type="Italic">Escherichia coli</Emphasis> (ETEC)

Type I fimbriae commonly expressed by Escherichia coli mediate initial attachment of bacteria to host epithelial cells. However, the role of type I fimbriae in the adherence of porcine enterotoxigenic E. coli (ETEC) to host receptors is unclear. In this study, we examined the role of type I fimbriae in the adherence and biofilm formation of F18ac+ ETEC by constructing mutant strains with deletion of type I fimbrial major subunit (fimA) or minor subunit (fimH). The data indicated that the isogenic ΔfimA and ΔfimH mutants showed significantly lower adherence to porcine epithelial IPEC-1 and IPEC-J2 cells as compared to the F18ac+ ETEC parent strain. In addition, the adherence of F18ac+ ETEC to both cell lines was blocked by the presence of 0.5% D-mannose in the cell culture medium. In addition, both mutant strains impaired their ability to form biofilm in vitro. Interestingly, the deletion of fimA or fimH genes resulted in remarkable up-regulation of the expression of adhesin involved in diffuse adherence (AIDA-I). These results indicated that type I fimbriae may be required for efficient adherence of F18ac+ ETEC to pig epithelial cells and, perhaps, biofilm formation.     

Denitrification by Chromobacterium violaceum

One host (Rana catesbiana)-associated and two free-living mesophilic strains of bacteria with violet pigmentation and biochemical characteristics of Chromobacterium violaceum were isolated from freshwater habitats. Cells of each freshly isolated strain and of strain ATCC 12472 (the neotype strain) grew anaerobically with glucose as the sole carbon and energy source. The major fermentation products of cells grown in Trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.) supplemented with glucose included acetate, small amounts of propionate, lactate, and pyruvate. The final cell yield and culture growth rate of each strain cultured anaerobically in this medium increased approximately twofold with the addition of 2 mM NaNO₃. Final growth yields increased in direct proportion to the quantity of added NaNO₃ over the range of 0.5 to 5 mM. Each strain reduced NO₃⁻, producing NO₂⁻, NO, and N₂O. NO₂⁻ accumulated transiently. With 2 mM NaNO₃ in the medium, N₂O made up 85 to 98% of the N product recovered with each strain. N-oxides were recovered in the same quantity and distribution whether 0.01 atm (ca. 1 kPa) of C₂H₂ (added to block N₂O reduction) was present or not. Neither N₂ production nor gas accumulation was detected during NO₃⁻ reduction by growing cells. Cell growth in media containing 0.5 to 5 mM NaNO₂ in lieu of NaNO₃ was delayed, and although N₂O was produced by the end of growth, NO₂⁻ -containing media did not support growth to an extent greater than did medium lacking NO₃⁻ or NO₂⁻. The data indicate that C. violaceum cells ferment glucose or denitrify, terminating denitrification with the production of N₂O, and that NO₂⁻ reduction to N₂O is not coupled to growth but may serve as a detoxification mechanism. No strain detectably fixed N₂ (reduced C₂H₂).     

Mutation and Selection of Lactobacillus plantarum Strains That Do Not Produce Carbon Dioxide from Malate

A differential medium was developed to distinguish between malate-decarboxylating (MDC⁺) and -non-decarboxylating (MDC⁻) strains of Lactobacillus plantarum. MDC⁻ strains produced a visible acid reaction in the medium, whereas MDC⁺ strains did not. Use of the medium allowed for rapid screening and isolation of mutagenized cells that had lost the ability to produce CO₂ from malate.     

Ae4 (Slc4a9) Anion Exchanger Drives Cl? Uptake-dependent Fluid Secretion by Mouse Submandibular Gland Acinar Cells

Transcellular Cl⁻ movement across acinar cells is the rate-limiting step for salivary gland fluid secretion. Basolateral Nkcc1 Na⁺-K⁺-2Cl⁻ cotransporters play a critical role in fluid secretion by promoting the intracellular accumulation of Cl⁻ above its equilibrium potential. However, salivation is only partially abolished in the absence of Nkcc1 cotransporter activity, suggesting that another Cl⁻ uptake pathway concentrates Cl⁻ ions in acinar cells. To identify alternative molecular mechanisms, we studied mice lacking Ae2 and Ae4 Cl⁻/HCO₃⁻ exchangers. We found that salivation stimulated by muscarinic and β-adrenergic receptor agonists was normal in the submandibular glands of Ae2^−/− mice. In contrast, saliva secretion was reduced by 35% in Ae4^−/− mice. The decrease in salivation was not related to loss of Na⁺-K⁺-2Cl⁻ cotransporter or Na⁺/H⁺ exchanger activity in Ae4^−/− mice but correlated with reduced Cl⁻ uptake during β-adrenergic receptor activation of cAMP signaling. Direct measurements of Cl⁻/HCO₃⁻ exchanger activity revealed that HCO₃⁻-dependent Cl⁻ uptake was reduced in the acinar cells of Ae2^−/− and Ae4^−/− mice. Moreover, Cl⁻/HCO₃⁻ exchanger activity was nearly abolished in double Ae4/Ae2 knock-out mice, suggesting that most of the Cl⁻/HCO₃⁻ exchanger activity in submandibular acinar cells depends on Ae2 and Ae4 expression. In conclusion, both Ae2 and Ae4 anion exchangers are functionally expressed in submandibular acinar cells; however, only Ae4 expression appears to be important for cAMP-dependent regulation of fluid secretion.     

Shift in Phenotypic Characteristics of Enterotoxigenic Escherichia coli (ETEC) Isolated from Diarrheal Patients in Bangladesh

Background

Enterotoxigenic Escherichia coli (ETEC) is one of the most common causes of bacterial diarrhea. Over the last decade, from 1996 to 2012, changes in the virulence antigen properties of ETEC such as heat labile (LT) and heat stable (ST) toxins, colonization factors (CFs), and ‘O’-serogroups have been observed. The aim of this prospective study was to compare changes in antigenic profiles of ETEC strains isolated from a 2% surveillance system at the icddr,b hospital in Dhaka, Bangladesh between 2007–2012 and an earlier time period of 1996–1998 conducted at the same surveillance site.

Methodology

In the surveillance system every 50^th patient attending the hospital was screened for major enteric pathogens including ETEC, Vibrio cholerae, Shigella spp. and Salmonella spp. from January 2007 to December 2012.

Principal Findings

Of the 15,152 diarrheal specimens tested between 2007–2012, the overall rate of ETEC isolation was 11%; of these, 43% were LT/ST, 27% LT and 30% ST positive. Isolation rate of ST-ETEC (p<0.009) and LT/ST ETEC (p<0.011) during 2007–2012 period differed significantly compared to those seen between 1996–1998. In comparison to the 1996–1998 period, difference in CF profile of ETEC isolates during 2007–2012 was observed particularly for strains expressing CS7 (12.4%), CS14 (9.5%) and CS17 (10.0%). The predominant CF types were CS5+CS6, CFA/I, CS7, CS17, CS1+CS3, CS6 and CS14. The most common serogroups among the CF positive ETEC isolates were O115, O114, O6, O25 and O8. A strong association was found between CFs and ‘O’ serogroups i.e. between CS5+CS6 and (O115 and O126); CS7 and (O114), CFA/I and (O78 and O126), CS17 and (O8 and O167) and CS1/CS2+CS3 and (O6).

Conclusion

The analyses show a shift in prevalence of antigenic types of ETEC over the study period; the information is important in designing effective ETEC vaccines with broad protective coverage.     

Deletion of IL-33R (ST2) Abrogates Resistance to EAE in BALB/C Mice by Enhancing Polarization of APC to Inflammatory Phenotype

The administration of interleukin 33 and deletion of IL-33 receptor, ST2 molecule, affects the induction of autoimmunity in different experimental models of human autoimmune diseases. The aim of this study was to analyze the effect of ST2 deletion on the induction of experimental autoimmune encephalomyelitis (EAE) in resistant BALB/c mice. Mice were immunized with MOG_35–55 peptide or disease was induced by passive transfer of encephalitogenic singenic cells and EAE was clinically and histologically evaluated. Expression of intracellular inflammatory cytokines, markers of activation and chemokine receptors on lymphoid tissue and CNS infiltrating mononuclear cells was analyzed by flow cytometry. We report here that deletion of ST2^−/− molecule abrogates resistance of BALB/c mice to EAE induction based on clinical and histopathological findings. Brain and spinal cord infiltrates of ST2^−/− mice had significantly higher number of CD4⁺ T lymphocytes containing inflammatory cytokines compared to BALB/c WT mice. Adoptive transfer of ST2^−/− primed lymphocytes induced clinical signs of the disease in ST2^−/− as well as in WT mice. MOG_35–55 restimulated ST2^−/− CD4⁺ cells as well as ex vivo analyzed lymph node cells had higher expression of T-bet and IL-17, IFN-γ, TNF-α and GM-CSF in comparison with WT CD4⁺ cells. ST2^−/− mice had higher percentages of CD4⁺ cells expressing chemokine receptors important for migration to CNS in comparison with WT CD4⁺ cells. Draining lymph nodes of ST2^−/− mice contained higher percentage of CD11c⁺CD11b⁺CD8⁻ cells containing inflammatory cytokines IL-6 and IL-12 with higher expression of activation markers. Transfer of ST2^−/− but not WT dendritic cells induced EAE in MOG_35–55 immunized WT mice. Our results indicate that ST2 deficiency attenuates inherent resistance of BALB/c mice to EAE induction by enhancing differentiation of proinflammatory antigen presenting cells and consecutive differentiation of encephalitogenic T cells in the draining lymph node rather than affecting their action in the target tissue.     

Arrestins function in cAR1 GPCR-mediated signaling and cAR1 internalization in the development of Dictyostelium discoideum

Oscillation of chemical signals is a common biological phenomenon, but its regulation is poorly understood. At the aggregation stage of Dictyostelium discoideum development, the chemoattractant cAMP is synthesized and released at 6-min intervals, directing cell migration. Although the G protein–coupled cAMP receptor cAR1 and ERK2 are both implicated in regulating the oscillation, the signaling circuit remains unknown. Here we report that D. discoideum arrestins regulate the frequency of cAMP oscillation and may link cAR1 signaling to oscillatory ERK2 activity. Cells lacking arrestins (adcB⁻C⁻) display cAMP oscillations during the aggregation stage that are twice as frequent as for wild- type cells. The adcB⁻C⁻ cells also have a shorter period of transient ERK2 activity and precociously reactivate ERK2 in response to cAMP stimulation. We show that arrestin domain–containing protein C (AdcC) associates with ERK2 and that activation of cAR1 promotes the transient membrane recruitment of AdcC and interaction with cAR1, indicating that arrestins function in cAR1-controlled periodic ERK2 activation and oscillatory cAMP signaling in the aggregation stage of D. discoideum development. In addition, ligand-induced cAR1 internalization is compromised in adcB⁻C⁻ cells, suggesting that arrestins are involved in elimination of high-affinity cAR1 receptors from cell surface after the aggregation stage of multicellular development.     

Cold Acclimation in Arabidopsis thaliana

The abilities of two races of Arabidopsis thaliana L. (Heyn), Landsberg erecta and Columbia, to cold harden were examined. Landsberg, grown at 22 to 24°C, increased in freezing tolerance from an initial 50% lethal temperature (LT₅₀) of about −3°C to an LT₅₀ of about −6°C after 24 hours at 4°C; LT₅₀ values of −8 to −10°C were achieved after 8 to 9 days at 4°C. Similar increases in freezing tolerance were obtained with Columbia. In vitro translation of poly(A⁺) RNA isolated from control and cold-treated Columbia showed that low temperature induced changes in the population of translatable mRNAs. An mRNA encoding a polypeptide of about 160 kilodaltons (isoelectric point about 4.5) increased markedly after 12 to 24 h at 4°C, as did mRNAs encoding four polypeptides of about 47 kilodaltons (isoelectric points ranging from 5-5.5). Incubation of Columbia callus tissue at 4°C also resulted in increased levels of the mRNAs encoding the 160 kilodalton polypeptide and at least two of the 47 kilodalton polypeptides. In vivo labeling experiments using Columbia plants and callus tissue indicated that the 160 kilodalton polypeptide was synthesized in the cold and suggested that at least two of the 47 kilodalton polypeptides were produced. Other differences in polypeptide composition were also observed in the in vivo labeling experiments, some of which may be the result of posttranslational modifications of the 160 and 47 kilodalton polypeptides.