Amoebicidal activity of Cassia angustifolia extract and its effect on Acanthamoeba triangularis autophagy-related gene expression at the transcriptional level.

Cassia angustifolia Vahl. plant is used for many therapeutic purposes, for example, in people with constipation, skin diseases, including helminthic and parasitic infections. In our study, we demonstrated an amoebicidal activity of C. angustifolia extract against Acanthamoeba triangularis trophozoite at a micromolar level. Scanning electron microscopy (SEM) images displayed morphological changes in the Acanthamoeba trophozoite, which included the formation of pores in cell membrane and the membrane rupture. In addition to the amoebicidal activity, effects of the extract on surviving trophozoites were observed, which included cyst formation and vacuolization by a microscope and transcriptional expression of Acanthamoeba autophagy in response to the stress by quantitative polymerase chain reaction. Our data showed that the surviving trophozoites were not transformed into cysts and the trophozoite number with enlarged vacuole was not significantly different from that of untreated control. Molecular analysis data demonstrated that the mRNA expression of AcATG genes was slightly changed. Interestingly, AcATG16 decreased significantly at 12 h post treatment, which may indicate a transcriptional regulation by the extract or a balance of intracellular signalling pathways in response to the stress, whereas AcATG3 and AcATG8b remained unchanged. Altogether, these data reveal the anti-Acanthamoeba activity of C. angustifolia extract and the autophagic response in the surviving trophozoites under the plant extract pressure, along with data on the formation of cysts. These represent a promising plant for future drug development. However, further isolation and purification of an active compound and cytotoxicity against human cells are needed, including a study on the autophagic response at the protein level.

Investigating selected host and parasite factors potentially impacting upon seasonal malaria chemoprevention in Bama, Burkina Faso.

BACKGROUND: Since 2014, seasonal malaria chemoprevention (SMC) with amodiaquine-sulfadoxine-pyrimethamine (AQ-SP) has been implemented on a large scale during the high malaria transmission season in Burkina Faso. This paper reports the prevalence of microscopic and submicroscopic malaria infection at the outset and after the first round of SMC in children under 5 years old in Bama, Burkina Faso, as well as host and parasite factors involved in mediating the efficacy and tolerability of SMC. METHODS: Two sequential cross-sectional surveys were conducted in late July and August 2017 during the first month of SMC in a rural area in southwest Burkina Faso. Blood smears and dried blood spots were collected from 106 to 93 children under five, respectively, at the start of SMC and again 3 weeks later. Malaria infection was detected by microscopy and by PCR from dried blood spots. For all children, day 7 plasma concentrations of desethylamodiaquine (DEAQ) were measured and CYP2C8 genetic variants influencing AQ metabolism were genotyped. Samples were additionally genotyped for pfcrt K76T and pfmdr1 N86Y, molecular markers associated with reduced amodiaquine susceptibility. RESULTS: 2.8% (3/106) of children were positive for Plasmodium falciparum infection by microscopy and 13.2% (14/106) by nested PCR within 2 days of SMC administration. Three weeks after SMC administration, in the same households, 4.3% (4/93) of samples were positive by microscopy and 14.0% (13/93) by PCR (p = 0.0007). CYP2C8*2, associated with impaired amodiaquine metabolism, was common with an allelic frequency of 17.1% (95% CI 10.0-24.2). Day 7 concentration of DEAQ ranged from 0.48 to 362.80 ng/mL with a median concentration of 56.34 ng/mL. Pfmdr1 N86 predominated at both time points, whilst a non-significant trend towards a higher prevalence of pfcrt 76T was seen at week 3. CONCLUSION: This study showed a moderate prevalence of low-level malaria parasitaemia in children 3 weeks following SMC during the first month of administration. Day 7 concentrations of the active DEAQ metabolite varied widely, likely reflecting variability in adherence and possibly metabolism. These findings highlight factors that may contribute to the effectiveness of SMC in children in a high transmission setting.

Trichostatin A effects on gene expression in the protozoan parasite Entamoeba histolytica.

BACKGROUND: Histone modification regulates chromatin structure and influences gene expression associated with diverse biological functions including cellular differentiation, cancer, maintenance of genome architecture, and pathogen virulence. In Entamoeba, a deep-branching eukaryote, short chain fatty acids (SCFA) affect histone acetylation and parasite development. Additionally, a number of active histone modifying enzymes have been identified in the parasite genome. However, the overall extent of gene regulation tied to histone acetylation is not known. RESULTS: In order to identify the genome-wide effects of histone acetylation in regulating E. histolytica gene expression, we used whole-genome expression profiling of parasites treated with SCFA and Trichostatin A (TSA). Despite significant changes in histone acetylation patterns, exposure of parasites to SCFA resulted in minimal transcriptional changes (11 out of 9,435 genes transcriptionally regulated). In contrast, exposure to TSA, a more specific inhibitor of histone deacetylases, significantly affected transcription of 163 genes (122 genes upregulated and 41 genes downregulated). Genes modulated by TSA were not regulated by treatment with 5-Azacytidine, an inhibitor of DNA-methyltransferase, indicating that in E. histolytica the crosstalk between DNA methylation and histone modification is not substantial. However, the set of genes regulated by TSA overlapped substantially with genes regulated during parasite development: 73/122 genes upregulated by TSA exposure were upregulated in E. histolytica cysts (p-value = 6 x 10(-53)) and 15/41 genes downregulated by TSA exposure were downregulated in E. histolytica cysts (p-value = 3 x 10(-7)). CONCLUSION: This work represents the first genome-wide analysis of histone acetylation and its effects on gene expression in E. histolytica. The data indicate that SCFAs, despite their ability to influence histone acetylation, have minimal effects on gene transcription in cultured parasites. In contrast, the effect of TSA on E. histolytica gene expression is more substantial and includes genes involved in the encystation pathway. These observations will allow further dissection of the effects of histone acetylation and the genetic pathways regulating stage conversion in this pathogenic parasite.

Growth of the protozoan parasite Entamoeba histolytica in 5-azacytidine has limited effects on parasite gene expression.

BACKGROUND: In higher eukaryotes DNA methylation regulates important biological functions including silencing of gene expression and protection from adverse effects of retrotransposons. In the protozoan parasite Entamoeba histolytica, a DNA methyltransferase has been identified and treatment with 5-azacytidine (5-AzaC), a potent inhibitor of DNA methyltransferase, has been reported to attenuate parasite virulence. However, the overall extent of DNA methylation and its subsequent effects on global gene expression in this parasite are currently unknown. RESULTS: In order to identify the genome-wide effects of DNA methylation in E. histolytica, we used a short oligonucleotide microarray representing 9,435 genes (approximately 95% of all annotated amebic genes) and compared the expression profile of E. histolytica HM-1:IMSS parasites with those treated with 23 microM 5-AzaC for up to one week. Overall, 2.1% of genes tested were transcriptionally modulated under these conditions. 68 genes were upregulated and 131 genes down regulated (2-fold change; p-value < 0.05). Sodium-bisulfite treatment and sequencing of genes indicated that there were at least two subsets of genes with genomic DNA methylation in E. histolytica: (i) genes that were endogenously silenced by genomic DNA methylation and for which 5-AzaC treatment induced transcriptional de-repression, and (ii) genes that have genomic DNA methylation, but which were not endogenously silenced by the methylation. We identified among the genes down regulated by 5-AzaC treatment a cysteine proteinase (2.m00545) and lysozyme (52.m00148) both of which have known roles in amebic pathogenesis. Decreased expression of these genes in the 5-AzaC treated E. histolytica may account in part for the parasites reduced cytolytic abilities. CONCLUSION: This work represents the first genome-wide analysis of DNA-methylation in Entamoeba histolytica and indicates that DNA methylation has relatively limited effects on gene expression in this parasite.

Distamycin A selectively inhibits Acanthamoeba RNA synthesis and differentiation.

The effects of distamycin A on Acanthamoeba transcription, growth and differentiation were determined. Distamycin A inhibits transcription both in vitro and in vivo and can displace from DNA the transcription activator TATA binding protein promoter binding factor (TPBF). Inhibition in vivo is surprisingly selective for large rRNA precursors, 5S rRNA, profilin, S-adenosylmethionine synthetase, and extendin. Transcription from the TATA binding protein (TBP), TPBF, protein disulfide isomerase, tubulin and RNA polymerase II large subunit genes is only slightly inhibited. Moreover the rate of 5S rRNA transcription eventually recovers and exceeds that of untreated cells, while profilin transcription remains inhibited. Distamycin A inhibition is accompanied by a complex pattern of alterations to steady state levels of mRNAs. Actin, profilin and S-adenosylmethionine synthetase mRNAs are degraded, whereas mRNA encoding TBP is increased slightly in abundance. Transcription inhibition is accompanied by cessation of growth and severe morphological changes to Acanthamoeba, which are consistent with loss of production of mRNA encoding cytoskeletal proteins. Distamycin A also prevents starvation-induced differentiation of Acanthamoeba, in part due to complete prevention of cellulose production and cell wall formation.

Transcription of the Dictyostelium glycogen phosphorylase-2 gene is induced by three large promoter domains.

The promoter of the Dictyostelium glycogen phosphorylase-2 (gp2) gene possesses a profound AT-bias, typical of promoters in this organism. To understand how Dictyostelium achieves specificity during transcriptional regulation under the constraint of this highly biased nucleotide composition, we have documented the changes in chromatin structure associated with developmental induction of gp2 gene expression. DNase I hypersensitive analyses indicated the presence of several developmentally regulated nuclease-sensitive sites located upstream of the start codon: two strong sites at approximately -250 bp and -350 bp and three substantially weaker sites at -290 bp, -445 bp, and -505 bp. In vitro footprint analyses using nuclear extracts derived from several stages of development (corresponding to varying levels of gp2 expression) revealed three large regions of occupation that were developmentally regulated and corresponded to these nuclease-sensitive sites: -227 to -294 bp (domain 1), -327 to -383 bp (domain 2), and -416 to -534 bp (domain 3). The presence and the extent of the three regulatory domains was confirmed by in vivo footprint analyses spanning the same developmental time points. Southwestern analyses using probes encompassing these footprints demonstrated that probes corresponding to domains 1 and 3 both interacted with 83 and 77 kDa peptides. The domain 3 probe also interacted with a 92 kDa peptide, while only a 62 kDa peptide is recognized by the domain 2 probe. In all cases, peptides capable of binding these probes were found in nuclear extracts derived from differentiated cells and not in undifferentiated cell nuclear extract. Using nuclear extract from differentiated cells and probes corresponding to the three domains, gel mobility shift analyses detected ladders of retarded bands for both domains 1 and 3 and three major retarded bands for domain 2. These results suggest that specificity in transcriptional activation in the AT-rich promoters of Dictyostelium may be achieved by requiring multiple protein-DNA and/or protein-protein interactions to occur before induction can proceed.

Expression of Dictyostelium early gene, dutA, is independent of cAMP pulses but dependent on protein kinase A.

The untranslatable, RNA polymerase II-dependent gene (dutA) of Dictyostelium discoideum is induced early in development. However, unlike other early genes, dutA induction was not affected by cAMP pulses and occurred normally in various cAMP-related mutant cells, the results indicating that this induction depended solely on factors other than cAMP. In the knockout strain of the catalytic subunit of protein kinase A, dutA expression was severely blocked and not recovered by cAMP pulses. This demonstrates that even the cAMP-independent gene, dutA, requires protein kinase A for its expression.

Cloning and characterization of the G-box binding factor, an essential component of the developmental switch between early and late development in Dictyostelium.

During Dictyostelium development, the cAMP-regulated induction of cell-type-specific late genes marks a developmental switch from the initial formation of the multicellular organism to the differentiation of the various cell types that mediate morphogenesis and eventually give rise to the mature fruting body. The G-box binding factor (GBF) is a developmentally regulated Dictyostelium transcription factor whose affinity for a DNA sequence correlates with the ability of that sequence to confer inducibility to late gene promoters in response to high, continuous levels of extracellular cAMP. We report the purification of GBF and cloning of the gene that encodes it, as confirmed by in vitro production of GBF activity. The predicted protein is highly basic and contains two putative zinc fingers. Disruption of the GBF gene by homologous recombination results in the loss of all GBF DNA-binding activity, developmental arrest at the loose aggregate stage, and the loss of late gene induction during development or in response to extracellular cAMP. Constitutive expression of GBF complements the null phenotype and allows for the rapid activation of a class of late genes in response to cAMP. Our results indicate that GBF acts as an extracellular cAMP-responsive transcriptional activator regulating late gene expression and is an essential component of a developmental switch between aggregation and cellular morphogenesis.