Characterization of Entamoeba fatty acid elongases; validation as targets and provision of promising leads for new drugs against amebiasis.

Entamoeba histolytica is a protozoan parasite belonging to the phylum Amoebozoa that causes amebiasis, a global public health problem. E. histolytica alternates its form between a proliferative trophozoite and a dormant cyst. Trophozoite proliferation is closely associated with amebiasis symptoms and pathogenesis whereas cysts transmit the disease. Drugs are available for clinical use; however, they have issues of adverse effects and dual targeting of disease symptoms and transmission remains to be improved. Development of new drugs is therefore urgently needed. An untargeted lipidomics analysis recently revealed structural uniqueness of the Entamoeba lipidome at different stages of the parasite's life cycle involving very long (26-30 carbons) and/or medium (8-12 carbons) acyl chains linked to glycerophospholipids and sphingolipids. Here, we investigated the physiology of this unique acyl chain diversity in Entamoeba, a non-photosynthetic protist. We characterized E. histolytica fatty acid elongases (EhFAEs), which are typically components of the fatty acid elongation cycle of photosynthetic protists and plants. An approach combining genetics and lipidomics revealed that EhFAEs are involved in the production of medium and very long acyl chains in E. histolytica. This approach also showed that the K3 group herbicides, flufenacet, cafenstrole, and fenoxasulfone, inhibited the production of very long acyl chains, thereby impairing Entamoeba trophozoite proliferation and cyst formation. Importantly, none of these three compounds showed toxicity to a human cell line; therefore, EhFAEs are reasonable targets for developing new anti-amebiasis drugs and these compounds are promising leads for such drugs. Interestingly, in the Amoebazoan lineage, gain and loss of the genes encoding two different types of fatty acid elongase have occurred during evolution, which may be relevant to parasite adaptation. Acyl chain diversity in lipids is therefore a unique and indispensable feature for parasitic adaptation of Entamoeba.

A rare case of extraintestinal amebiasis.

BACKGROUND: Amoebiasis is caused by the protozoan Entamoeba histolytica, which is a rare infectious disease in developed countries. If the trophozoites enter the blood, it can spread through the body, such as brain, and lungs. Cases of simultaneous infection of multiple organs are extremely rare. CASE PRESENTATION: Here we report a case of simultaneous infection of amoeba in pulmonary pleura, urinary system and central nervous system. Although the patient received anti amoeba treatment, the prognosis of the patient was poor. CONCLUSIONS: In this patient, multiple extraintestinal amebic infections in the absence of clinically confirmed intestinal amebiasis or amebic liver abscess are rare and pose diagnostic challenges. The disseminated amebiasis has significantly increased the mortality. Early diagnosis and appropriate treatment may reduce the mortality of disseminated amebiasis.

Management of Entamoeba histolytica in the non-human primates at the Singapore Zoo.

Amebic dysentery caused by Entamoeba histolytica accounts for significant morbidity in the non-human primates (NHP) at the Singapore Zoo. This includes the animals in the collection as well as a sizeable free-roaming wild crab-eating macaque (Macaca fascicularis) population. The disease is of great concern because of its zoonotic potential. Passive surveillance, both ante and post-mortem, of NHP displaying clinical symptoms and active surveillance of NHP assessed to be at a higher risk of infection were carried out via fecal real-time polymerase chain reaction (PCR) testing for 4 years. Treatment of the disease with 25 mg/kg metronidazole BID for 10 days followed by 15 mg/kg paromomycin BID for 7 days achieved good clinical resolution in most cases that tested positive. Three diseased NHP with severe clinical signs of weight loss, lethargy, and diarrhea were anesthetized for veterinary diagnostic investigation. Mesenteric lymphadenopathy was consistently seen on ultrasound examination in these severe cases of entamoebiasis. Two animals eventually died of severe chronic enteritis due to the disease. The eradication of entamoebiasis in the NHP at the Singapore Zoo may be complicated by the maintenance of a disease reservoir in wildlife, but a combination of timely treatment and efforts at maintaining biosecurity can help manage the disease in the collection.

Escherichia coli mediated resistance of Entamoeba histolytica to oxidative stress is triggered by oxaloacetate.

Amebiasis, a global intestinal parasitic disease, is due to Entamoeba histolytica. This parasite, which feeds on bacteria in the large intestine of its human host, can trigger a strong inflammatory response upon invasion of the colonic mucosa. Whereas information about the mechanisms which are used by the parasite to cope with oxidative and nitrosative stresses during infection is available, knowledge about the contribution of bacteria to these mechanisms is lacking. In a recent study, we demonstrated that enteropathogenic Escherichia coli O55 protects E. histolytica against oxidative stress. Resin-assisted capture (RAC) of oxidized (OX) proteins coupled to mass spectrometry (OX-RAC) was used to investigate the oxidation status of cysteine residues in proteins present in E. histolytica trophozoites incubated with live or heat-killed E. coli O55 and then exposed to H2O2-mediated oxidative stress. We found that the redox proteome of E. histolytica exposed to heat-killed E. coli O55 is enriched with proteins involved in redox homeostasis, lipid metabolism, small molecule metabolism, carbohydrate derivative metabolism, and organonitrogen compound biosynthesis. In contrast, we found that proteins associated with redox homeostasis were the only OX-proteins that were enriched in E. histolytica trophozoites which were incubated with live E. coli O55. These data indicate that E. coli has a profound impact on the redox proteome of E. histolytica. Unexpectedly, some E. coli proteins were also co-identified with E. histolytica proteins by OX-RAC. We demonstrated that one of these proteins, E. coli malate dehydrogenase (EcMDH) and its product, oxaloacetate, are key elements of E. coli-mediated resistance of E. histolytica to oxidative stress and that oxaloacetate helps the parasite survive in the large intestine. We also provide evidence that the protective effect of oxaloacetate against oxidative stress extends to Caenorhabditis elegans.

Case report: multiple and atypical amoebic cerebral abscesses resistant to treatment.

BACKGROUND: The parasite Entamoeba histolytica is the causal agent of amoebiasis, a worldwide emerging disease. Amebic brain abscess is a form of invasive amebiasis that is both rare and frequently lethal. This condition always begins with the infection of the colon by E. histolytica trophozoites, which subsequently travel through the bloodstream to extraintestinal tissues. CASE PRESENTATION: We report a case of a 71-year-old female who reported an altered state of consciousness, disorientation, sleepiness and memory loss. She had no history of hepatic or intestinal amoebiasis. A preliminary diagnosis of colloidal vesicular phase neurocysticercosis was made based on nuclear magnetic resonance imaging (NMRI). A postsurgery immunofluorescence study was positive for the 140 kDa fibronectin receptor of E. histolytica, although a serum analysis by ELISA was negative for IgG antibodies against this parasite. A specific E. histolytica 128 bp rRNA gene was identified by PCR in biopsy tissue. The final diagnosis was cerebral amoebiasis. The patient underwent neurosurgery to eliminate amoebic abscesses and was then given a regimen of metronidazole, ceftriaxone and dexamethasone for 4 weeks after the neurosurgery. However, a rapid decline in her condition led to death. CONCLUSIONS: The present case of an individual with a rare form of cerebral amoebiasis highlights the importance of performing immunofluorescence, NMRI and PCR if a patient has brain abscess and a poorly defined diagnosis. Moreover, the administration of corticosteroids to such patients can often lead to a rapid decline in their condition.

Intestinal parasites including Cryptosporidium, Cyclospora, Giardia, and Microsporidia, Entamoeba histolytica, Strongyloides, Schistosomiasis, and Echinococcus: Guidelines from the American Society of Transplantation Infectious Diseases Community of Practice.

These updated guidelines from the Infectious Diseases Community of Practice of the American Society of Transplantation review the diagnosis, prevention, and management of intestinal parasites in the pre- and post-transplant period. Intestinal parasites are prevalent in the developing regions of the world. With increasing travel to and from endemic regions, changing immigration patterns, and the expansion of transplant medicine in developing countries, they are increasingly recognized as a source of morbidity and mortality in solid-organ transplant recipients. Parasitic infections may be acquired from the donor allograft, from reactivation, or from de novo acquisition post-transplantation. Gastrointestinal multiplex assays have been developed; some of the panels include testing for Cryptosporidium, Cyclospora, Entamoeba histolytica, and Giardia, and the performance is comparable to conventional methods. A polymerase chain reaction test, not yet widely available, has also been developed to detect Strongyloides in stool samples. New recommendations have been developed to minimize the risk of Strongyloides donor-derived events. Deceased donors with epidemiological risk factors should be screened for Strongyloides and recipients treated if positive as soon as the results are available. New therapeutic agents and studies addressing the optimal treatment regimen for solid-organ transplant recipients are unmet needs.

All that glitters is not gold. A different cause for an "ulcerative colitis".

The authors present the case of a 32-year-old Caucasian male, engineer, who was submitted to a colonoscopy after a presumptive diagnosis of ulcerative colitis. The patient referred an acute bloody and mucous diarrhea, lasting for three weeks, with no fever or rectal tenesmus. Stool studies were negative. During the procedure, colonic segments with continuous hyperemic and exudative mucosa, with small papules with apical ulcers and erosions, were observed.

Amoebic liver abscess: an unusual cause for a right iliac fossa mass: a case report.

BACKGROUND: Amoebic liver abscess is the most common extra intestinal manifestation of amoebiasis in tropical countries. It usually presents with right hypochondrial pain, fever and anorexia. Amoebic liver abscess has gained clinical significance due to the wide variety of clinical presentations which can cause diagnostic dilemmas and high mortality in untreated cases. CASE PRESENTATION: We report a case of a 63-year-old male with a history of anorexia for 3 weeks, fever for 4 days and examination findings of tender hepatomegaly with a liver span of 15 cm in the mid clavicular line and a firm irregular mass in the right iliac fossa. Ultrasound scan of the abdomen showed two large liver abscesses with one of them leaking into the peritoneal cavity causing a localized pus collection, which had been walled off in the right iliac fossa. He was treated with metronidazole and liver abscesses were drained percutaneously under ultrasound scan guidance. The diagnosis of Entamoeba histolytica infection was confirmed with the serology and subsequently by PCR from the aspirated material. He made an uneventful recovery with resolution of the symptoms and right iliac fossa mass. CONCLUSION: Recognition of variable presentation of amoebic liver abscess is vital, considering the curable nature of this disease and potentially fatal outcome of untreated abscess. An intra-abdominal mass in a patient with amoebic liver abscess should raise the suspicion of a localized collection of pus and impending generalized peritonitis. Early diagnosis and prompt intervention can prevent the dreaded complication of peritonitis and toxemia, and hence reduce the consequent morbidity and mortality.

Metronidazole hydrazone conjugates: Design, synthesis, antiamoebic and molecular docking studies.

Metronidazole hydrazone conjugates (2-13) were synthesized and screened in vitro for antiamoebic activity against HM1: IMSS strain of Entamoeba histolytica. Six compounds were found to be better inhibitors of E. histolytica than the reference drug metronidazole. These compounds showed greater than 50-60% viability against HeLa cervical cancer cell line after 72 h treatment. Also, molecular docking study was undertaken on E. histolytica thioredoxin reductase (EhTHRase) protein which showed significant binding affinity in the active site. Out of the six actives, some of the compounds showed lipophilic characteristics.

[Ameboma: possible therapeutic decisions in an amebiasis-endemic region]. / Ameboma: posibles decisiones terapéuticas en región endémica de amibiasis.

INTRODUCTION: Amebiasis can mimic cecal tumors. Unless this infection is diagnosed in a timely manner, affected individuals may undergo extensive surgery. MATERIAL AND METHODS: We carried out a retrospective analytical study of the therapeutic approach to amebiasis in a second-level hospital in an area of central Mexico with a high prevalence of this infection. Records from 2005-2011 were reviewed. There were 261 cases of amebiasis. Twenty cases were diagnosed by the histopathologist or on the basis of serological results. Sixteen patients underwent surgery due to acute abdomen, and four received medical treatment with metronidazole. Three treatment groups were analyzed: 1. hemicolectomy, 2. appendicectomy and antiamebic therapy, and 3. antiamoebic therapy alone. In the non-surgical group, imaging studies showed improvement with medical therapy. RESULTS: Length of hospital stay was higher in the group undergoing extensive surgery (p < 0.0133). There were no statistically significant differences among the remaining variables. CONCLUSIONS: The incidence of ameboma in our environment is higher (7.6%) than that reported in the literature. We believe that, in endemic regions, ameboma should be ruled out in patients with a cecal mass. As part of the therapeutic approach, patients should be tested for amebiasis or receive antiamebic therapy with monitoring of the mass to avoid extensive resective surgery.

Hsp90 inhibitors as new leads to target parasitic diarrheal diseases.

Entamoeba histolytica and Giardia lamblia are anaerobic protozoan parasites that cause amebiasis and giardiasis, two of the most common diarrheal diseases worldwide. Current therapy relies on metronidazole, but resistance has been reported and the drug has significant adverse effects. Therefore, it is critical to search for effective, better-tolerated antiamebic and antigiardial drugs. We synthesized several examples of a recently reported class of Hsp90 inhibitors and evaluated these compounds as potential leads for antiparasitic chemotherapy. Several of these inhibitors showed strong in vitro activity against both E. histolytica and G. lamblia trophozoites. The inhibitors were rescreened to discriminate between amebicidal and giardicidal activity and general cytotoxicity toward a mammalian cell line. No mammalian cytotoxicity was found at >100 µM for 48 h for any of the inhibitors. To understand the mechanism of action, a competitive binding assay was performed using the fluorescent ATP analogue bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt) and recombinant E. histolytica Hsp90 preincubated in both the presence and absence of Hsp90 inhibitors. There was significant reduction in fluorescence compared to the level in the control, suggesting that E. histolytica Hsp90 is a selective target. The in vivo efficacy and safety of one Hsp90 inhibitor in a mouse model of amebic colitis and giardiasis was demonstrated by significant inhibition of parasite growth at a single oral dose of 5 mg/kg of body weight/day for 7 days and 10 mg/kg/day for 3 days. Considering the results for in vitro activity and in vivo efficacy, Hsp90 inhibitors represent a promising therapeutic option for amebiasis and giardiasis.

Src and PI3 K inhibitors affect the virulence factors of Entamoeba histolytica.

Protein kinases (PKs) of parasitic protozoa are being evaluated as drug targets. A large number of protein kinases within the protein kinome of Entamoeba histolytica strongly suggest that protein phosphorylation is a key component of pathogenesis regulation by this parasite. PI3 K and Src are kinases previously described in this parasite, but their role is poorly understood. Here, the effect of Src-1-inhibitor and PI3 K inhibitor (Wortmannin) on the virulence factors of E. histolytica was evaluated. Results show that both inhibitors affect the actin cytoskeleton and the amoebic movement. Also, the proteolytic activity is diminished by Wortmannin, but not by Src-inhibitor-1; however, the phagocytic capacity is diminished by Wortmannin and Src-1-inhibitor. Finally, we found that the virulence in vivo of E. histolytica is affected by Wortmannin but not by Src-1-inhibitor. This study opens the way for the design of anti-amoebic drugs based on kinase inhibition.

Entamoeba histolytica induced NETosis and the dual role of NETs in amoebiasis.

Entamoeba histolytica (Eh), a microaerophilic parasite, causes deadly enteric infections that result in Amoebiasis. Every year, the count of invasive infections reaches 50 million approximately and 40,000 to 1,00,000 deaths occurring due to amoebiasis are reported globally. Profound inflammation is the hallmark of severe amoebiasis which is facilitated by immune first defenders, neutrophils. Due to size incompatibility, neutrophils are unable to phagocytose Eh and thus, came up with the miraculous antiparasitic mechanism of neutrophil extracellular traps (NETs). This review provides an in-depth analysis of NETosis induced by Eh including the antigens involved in the recognition of Eh and the biochemistry of NET formation. Additionally, it underscores its novelty by describing the dual role of NETs in amoebiasis where it acts as a double-edged sword in terms of both clearing and exacerbating amoebiasis. It also provides a comprehensive account of the virulence factors discovered to date that are implicated directly and indirectly in the pathophysiology of Eh infections through the lens of NETs and can be interesting drug targets.

Inhibition of choline kinase as an antiamoebic approach in Entamoeba histolytica infection.

Entamoeba histolytica is the parasite responsible for amoebiasis, which can result in amoebic colitis or amoebic liver abscess. Metronidazole has been the conventional treatment for intestinal amoebiasis, but concerns regarding resistance have emerged due to the identification of resistance pathways in E. histolytica. This study investigates a novel anti-amoebic approach targeting the CDP-choline pathway. Inhibition studies were conducted using potential choline kinase (CK) inhibitors to inhibit the EhCK enzyme, and RNA interference was employed to knock down the EhCK gene. Km and Vmax of purified EhCK and hCKa2 proteins were determined by pyruvate kinase-lactate dehydrogenase (PK-LDH) coupled assay. The IC50 values for EhCK and hCKa2 were determined with several commercial CK inhibitors. Selected inhibitors were incubated with E. histolytica trophozoites for 48 hours to determine the EC50 for each inhibitor. Silencing of gene encoding EhCK was carried out using duplex siRNA and the gene expression level was measured by real-time qPCR. Based on the IC50 values, three of the inhibitors, namely CK37, flavopiridol and H-89 were more potent against EhCK than hCKa2. Trophozoites growth inhibition showed that only HDTAB, H-89 and control drug metronidazole could penetrate and induce cell death after 48-hour incubation. siRNA concentration of 10 µg/mL was used for the transfection of positive control GAPDH, EhCK, and non-targeting GFP siRNAs. RNAi experiment concluded with positive control GAPDH downregulated by 99% while the level of EhCK mRNA was downregulated by 47%. In this study, potential inhibitors of EhCK and siRNA have been identified, paving the way for further refinement and testing to enhance their potency against EhCK while sparing hCK. The utilization of these specific inhibitors and siRNA targeting EhCK represents a novel approach to impede the growth of E. histolytica by disrupting its phospholipid synthesis pathway.

Oral lactoferrin treatment resolves amoebic intracecal infection in C3H/HeJ mice.

Entamoeba histolytica is a protozoan parasite that causes amoebiasis, an illness that affects many people around the world. We have previously reported that lactoferrin is able to kill E. histolytica in in vitro cultures. The aim of the present study was to evaluate the therapeutic effect of orally administered bovine lactoferrin in the control of intestinal amoebiasis of susceptible C3H/HeJ mice. The results showed that 20 mg lactoferrin/kg orally administered each day for 1 week was able to eliminate the infection in 63% of the mice, since neither trophozoites nor evidence of epithelial damage and (or) swelling were found in tissue sections of the cecum. The rest of the treated animals (37%) showed a decrease in trophozoite numbers and mucus secreted to the lumen, as compared with untreated and infected mice (p < 0.05). By immunohistochemistry, the profile of secreted cytokines in the cecum revealed that infected but untreated animals showed a mixed Th1/regulatory cytokines profile, whereas the cecum of mice treated (cured) showed a Th2 cytokine profile (IL-4) and expression of the multifunctional IL-6. In addition, cytokines and increasing cecal production of total IgA antibodies were found associated with little inflammation and disease control observed in the cecum of lactoferrin-treated animals. These results suggest that oral administration of lactoferrin can control intestinal amoebic infection probably by killing amoebas or favoring their removal and reestablish the antiinflammatory intestinal environment.

Metronidazole thiosalicylate conjugates: synthesis, crystal structure, docking studies and antiamoebic activity.

Metronidazole thiosalicylate conjugates were synthesized and crystallised in order to discover new molecules having better efficacy than therapeutically administered drug metronidazole, used against Entamoeba histolytica. The three compounds (4-6) showed lower IC(50) values than metronidazole on HM1:IMSS strain of E. histolytica and displayed low cytotoxicity on MCF-7 cell line. In order to get an insight into the mechanisms of action of these compounds, a homology model of E. histolytica thioredoxin reductase (EhTHRase) was constructed and molecular docking was performed into the binding pocket to identify the nature of interactions. The docking studies suggest that the improved inhibitory activity of the newly synthesised metronidazole analogues could be due to involvement of the additional hydrophobic interactions in the binding mode. The result of the present study indicates the molecular fragments that play an essential role in improving the antiamoebic activity.