Seasonal prevalence, risk factors, and One Health intervention for prevention of intestinal parasitic infection in underprivileged communities on the Thai-Myanmar border.

BACKGROUND: Tha Song Yang District, located on the Thai-Myanmar border, contributes to the second highest cases of amoebic dysentery due to intestinal parasitic infections (IPI). However, there were limited disease prevalence data, specific surveillance systems, and interventions available. OBJECTIVE: This study aimed to explore the epidemiological features of the IPIs and apply the One Health (OH) approach to solve IPI-related problems. METHODS: Prevalence of asymptomatic infections in human and animals, yearly symptomatic cases, and associated risk factors were investigated. The OH intervention included improving the knowledge, attitude, and practice (KAP) of the community, microscopic diagnosis training, and stakeholder engagement for IPI prevention designs. RESULTS: The prevalence of asymptomatic cases was much higher than that of the symptomatic cases. Infective stages of the intestinal parasites were discovered in animal stool and water samples, indicating possible transmission routes. One year after the intervention, there were significant declines in asymptomatic IPIs and symptomatic cases of amoebic dysentery. Significant improvements in KAP and awareness regarding water and manure-waste management of the community were observed. CONCLUSION: We reported the successful application of the OH intervention in reducing the IPI prevalence and mitigating disease-related risks. The intervention might be applied to address other infectious diseases in the future.

Host-antibody inductivity of virulent Entamoeba histolytica and non-virulent Entamoeba moshkovskii in a mouse model.

BACKGROUND: Despite similarities in morphology, gene and protein profiles, Entamoeba histolytica and E. moshkovskii show profound differences in pathogenicity. Entamoeba histolytica infection might result in amoebic dysentery and liver abscess, while E. moshkovskii causes only mild diarrhea. Extensive studies focus on roles of host immune responses to the pathogenic E. histolytica; however, evidence for E. moshkovskii remains scarce. METHODS: To study differences in host-antibody response profiles between E. histolytica and E. moshkovskii, mice were immunized intraperitoneally with different sets of Entamoeba trophozoites as single species, mixed species and combinations. RESULTS: Mice prime-immunized with E. histolytica and E. moshkovskii combination, followed by individual species, exhibited higher IgG level than the single species immunization. Mice immunized with E. moshkovskii induced significantly higher levels and long-lasting antibody responses than those challenged with E. histolytica alone. Interestingly, E. histolytica-specific anti-sera promoted the cytopathic ability of E. histolytica toward Chinese hamster ovarian (CHO) cells, but showed no effect on cell adhesion. There was no significant effect of immunized sera on cytopathic activity and adhesion of E. moshkovskii toward both CHO and human epithelial human colonic (Caco-2) cell lines. Monoclonal-antibody (mAb) characterization demonstrated that 89% of E. histolytica-specific mAbs produced from mice targeted cytoplasmic and cytoskeletal proteins, whereas 73% of E. moshkovskii-specific mAbs targeted plasma membrane proteins. CONCLUSIONS: The present findings suggest that infection with mixed Entamoeba species or E. moshkovskii effectively induces an antibody response in mice. It also sheds light on roles of host antibody response in the pathogenic difference of E. histolytica and E. moshkovskii trophozoites, and cell surface protein modifications of the amoebic parasites to escape from host immune system.

Enhancement of insect susceptibility and larvicidal efficacy of Cry4Ba toxin by calcofluor.

BACKGROUND: Mosquitoes transmit many vector-borne infectious diseases including malaria, dengue, chikungunya, yellow fever, filariasis, and Japanese encephalitis. The insecticidal δ-endotoxins Cry4, Cry11, and Cyt produced from Bacillus thuringiensis have been used for bio-control of mosquito larvae. Cry δ-endotoxins are synthesised as inactive protoxins in the form of crystalline inclusions in which they are processed to active toxins in larval midgut lumen. Previously, we demonstrated that the activated Cry4Ba toxin has to alter the permeability of the peritrophic membrane (PM), allowing toxin passage across PM to reach specific receptors on microvilli of larval midgut epithelial cells, where the toxin undergoes conformational changes, followed by membrane insertion and pore formation, resulting in larval death. A peritrophic membrane (PM)-binding calcofluor has been proposed to inhibit chitin formation and enhance baculovirus infection of lepidopteran Trichoplusia ni. METHODS: In this study, Aedes aegypti larvae were fed with the calcofluor and Cry4Ba toxin to investigate the effect of this agent on the toxicity of the Cry4Ba toxin. RESULTS: Calcofluor displayed an enhancing effect when co-fed with the Cry4Ba wild-type toxin. The agent could restore the killing activity of the partially active Cry4Ba mutant E417A/Y455A toward Ae. aegypti larvae. PM destruction was observed after larval challenge with calcofluor together with the toxin. Interestingly, calcofluor increased Cry4Ba toxin susceptibility toward semi-susceptible Culex quinquefasciatus larvae. However, calcofluor alone or in combination with the toxin showed no mortality effect on non-susceptible fresh-water fleas, Moina macrocopa. CONCLUSIONS: Our results suggest that PM may contribute to the resistance of the mosquito larvae to Cry4Ba toxin. The PM-permeability alternating calcofluor might be a promising candidate for enhancing insect susceptibility, which will consequently improve Cry4Ba efficacy in field settings in the future.

Functional assembly of 260-kDa oligomers required for mosquito-larvicidal activity of the Bacillus thuringiensis Cry4Ba toxin.

Oligomerization has been shown to contribute to the toxicity of Bacillus thuringiensis Cry toxins. Mutations have been made in the Cry4Ba toxin and resulted in toxic to non-toxic mutants toward Aedes aegypti larvae. In this study, Cry4Ba wild type and mutants were analyzed for oligomer formation in vitro, biochemical properties and their relationships with larvicidal activity. In vitro, the Cry4Ba forms two-main types of the oligomers including (1) the 260-kDa and larger oligomers, which assembled in the carbonate buffer, pH 10.0 and completely dissociated by heating at 90°C and (2) 190-kDa oligomer, which was induced by heat, sodium-salt and detergent addition. Polar and charge residues in the toxin domain I and II may contribute to formation of the 260-kDa oligomers. A single Cys-525 in domain III was replaced with serine resulting in the C525S mutant, which exhibited a 50% reduction in larvicidal activity compared to the Cry4Ba wild-type. The mutant exhibited partial loss in larger oligomer of the 260kDa and total loss of 190-kDa oligomer. The results revealed an important role of the Cys-525 in intermolecular disulfide formation of larger oligomer as well as the 190-kDa oligomer. Despite of their formations in the receptor free condition, the 260-kDa and larger oligomers were found to strongly correspond to Cry4Ba toxicity suggesting their functional roles in the A. aegypti larvae. Also, possible roles of the 260-kDa and larger oligomers have been proposed in this report.

Sequence and apoptotic activity of VacA cytotoxin cloned from a Helicobacter pylori Thai clinical isolate.

The vacuolating cytotoxin VacA produced by Helicobacter pylori induces the formation of large cytoplasmic vacuoles in host gastric epithelial cells as well as a release of cytochrome C from mitochondria resulting in cell apoptosis. Considerable sequence diversity in VacA relating to different degrees of disease severity is observed with clinical samples from a multitude of geographic places. In this study we describe expression in Escherichia coli, purification to homogeneity and in vitro assay of its apoptotic activity of a VacA toxin from a H. pylori isolate of a Thai patient with gastrointestinal lymphoma. Sequencing revealed that the deduced amino acid sequence of the cloned Thai isolate VacA is similar to H. pylori s1/m2 type strains. The percent sequence similarity to the model strain 60190 was lower due to the presence of extra amino acids in the mid (m) region. The purified VacA toxin exhibited significant apoptotic activity on both T84 and MDCK epithelial cell lines, as revealed by DAPI staining, whereby the observed activity was significantly higher on MDCK cells. These findings could relate to a modulation of VacA activity on host cells in the Thai isolate-VacA toxin that may differ from those of the model strain.

Functional characterizations of residues Arg-158 and Tyr-170 of the mosquito-larvicidal Bacillus thuringiensis Cry4Ba.

The insecticidal activity of Bacillus thuringiensis (Bt) Cry toxins involves toxin stabilization, oligomerization, passage across the peritrophic membrane (PM), binding to midgut receptors and pore-formation. The residues Arg-158 and Tyr-170 have been shown to be crucial for the toxicity of Bt Cry4Ba. We characterized the biological function of these residues. In mosquito larvae, the mutants R158A/E/Q (R158) could hardly penetrate the PM due to a significantly reduced ability to alter PM permeability; the mutant Y170A, however, could pass through the PM, but degraded in the space between the PM and the midgut epithelium. Further characterization by oligomerization demonstrated that Arg-158 mutants failed to form correctly sized high-molecular weight oligomers. This is the first report that Arg-158 plays a role in the formation of Cry4Ba oligomers, which are essential for toxin passage across the PM. Tyr-170, meanwhile, is involved in toxin stabilization in the toxic mechanism of Cry4Ba in mosquito larvae.

Aedes aegypti membrane-bound alkaline phosphatase expressed in Escherichia coli retains high-affinity binding for Bacillus thuringiensis Cry4Ba toxin.

Glycosylphosphatidylinositol-linked alkaline phosphatase (GPI-ALP) from the epithelial membrane of the larval midgut of Aedes aegypti was previously identified as a functional receptor of the Bacillus thuringiensis Cry4Ba toxin. Here, heterologous expression in Escherichia coli of the cloned ALP, lacking the secretion signal and GPI attachment sequences, and assessment of its binding characteristics were further investigated. The 54-kDa His tag-fused ALP overexpressed as an inclusion body was soluble when phosphate buffer (pH 7.5) was supplemented with 8 M urea. After renaturation in a nickel-nitrilotriacetic acid (Ni-NTA) affinity column, the refolded ALP protein was able to retain its phosphatase activity. This refolded ALP also showed binding to the 65-kDa activated Cry4Ba toxin under nondenaturing (dot blot) conditions. Quantitative binding analysis using a quartz crystal microbalance revealed that the purified ALP immobilized on a gold electrode was bound by the Cry4Ba toxin in a stoichiometry of approximately 1:2 and with high affinity (dissociation constant [K(d)] of ∼14 nM) which is comparable to that calculated from kinetic parameters (dissociation rate constant [k(off)]/binding constant [k(on)]). Altogether, the data presented here of the E. coli-expressed ALP from A. aegypti retaining high-affinity toxin binding support our notion that glycosylation of this receptor is not required for binding to its counterpart toxin, Cry4Ba.

In vivo identification of Bacillus thuringiensis Cry4Ba toxin receptors by RNA interference knockdown of glycosylphosphatidylinositol-linked aminopeptidase N transcripts in Aedes aegypti larvae.

Bacillus thuringiensis Cry4Ba toxin selectively kills Aedes aegypti mosquito larvae as it is in part due to the presence of specific membrane-bound protein receptors. In this study, using data mining approach, we initially identified three potential glycosylphosphatidylinositol-linked aminopeptidase N (GPI-APN) isoforms, APN2778, APN2783 and APN5808, which are believed to act as Cry4Ba toxin receptors. These three isoforms that are functionally expressed in the larval midgut can be sequence-specific knocked down (ranging from ∼80 % to 95 %) by soaking the Aedes aegypti larvae in buffer of long double-stranded GPI-APN RNAs (∼300-680 bp). Finally, to see the physiological effect of APN knockdowns, the larvae were fed with Escherichia coli expressing Cry4Ba toxin. The results revealed that all the three identified GPI-APN isoforms may possibly function as a Cry4Ba receptor, particularly for APN2783 as those larvae with this transcript knockdown showed a dramatic increase in resistance to Cry4Ba toxicity.

Novel preparation and characterization of the alpha4-loop-alpha5 membrane-perturbing peptide from the Bacillus thuringiensis Cry4Ba delta-endotoxin.

Helices 4 and 5 of the Bacillus thuringiensis Cry4Ba delta-endotoxin have been shown to be important determinants for mosquito-larvicidal activity, likely being involved in membrane-pore formation. In this study, the Cry4Ba mutant protein containing an additional engineered tryptic cleavage site was used to produce the alpha4-alpha5 hairpin peptide by an efficient alternative strategy. Upon solubilization of toxin inclusions expressed in Escherichia coli and subsequent digestion with trypsin, the 130-kDa mutant protoxin was processed to protease-resistant fragments of ca. 47, 10 and 7 kDa. The 7-kDa fragment was identified as the alpha4-loop-alpha5 hairpin via N-terminal sequencing and mass spectrometry, and was successfully purified by size-exclusion FPLC and reversed-phase HPLC. Using circular dichroism spectroscopy, the 7-kDa peptide was found to exist predominantly as an alpha-helical structure. Membrane perturbation studies by using fluorimetric calcein-release assays revealed that the 7-kDa helical hairpin is highly active against unilamellar liposomes compared with the 65-kDa activated full-length toxin. These results directly support the role of the alpha4-loop-alpha5 hairpin in membrane perturbation and pore formation of the full-length Cry4Ba toxin.

Bacillus thuringiensis Cry4A and Cry4B mosquito-larvicidal proteins: homology-based 3D model and implications for toxin activity.

Three-dimensional (3D) models for the 65-kDa activated Cry4A and Cry4B delta-endotoxins from Bacillus thuringiensis subsp. israelensis that are specifically toxic to mosquito-larvae were constructed by homology modeling, based on atomic coordinates of the Cry1Aa and Cry3Aa crystal structures. They were structurally similar to the known structures, both derived 3D models displayed a three-domain organization: the N-terminal domain (I) is a seven-helix bundle, while the middle and C-terminal domains are primarily comprise of anti-parallel beta-sheets. Circular dichroism spectroscopy confirmed the secondary structural contents of the two homology-based Cry4 structures. A structural analysis of both Cry4 models revealed the following: (a) Residues Arg-235 and Arg-203 are located in the interhelical 5/6 loop within the domain I of Cry4A and Cry4B, respectively. Both are solvent exposed. This suggests that they are susceptible to tryptic cleavage. (b) The unique disulphide bond, together with a proline-rich region within the long loop connecting alpha4 and alpha5 of Cry4A, were identified. This implies their functional significance for membrane insertion. (c) Significant structural differences between both models were found within domain II that may reflect their different activity spectra. Structural insights from this molecular modeling study would therefore increase our understanding of the mechanic aspects of these two closely related mosquito-larvicidal proteins.