mRNA Polyadenylation Machineries in Intestinal Protozoan Parasites.

In humans, mRNA polyadenylation involves the participation of about 20 factors in four main complexes that recognize specific RNA sequences. Notably, CFIm25, CPSF73, and PAP have essential roles for poly(A) site selection, mRNA cleavage, and adenosine residues polymerization. Besides the relevance of polyadenylation for gene expression, information is scarce in intestinal protozoan parasites that threaten human health. To better understand polyadenylation in Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum, which represent leading causes of diarrhea worldwide, genomes were screened for orthologs of human factors. Results showed that Entamoeba histolytica and C. parvum have 16 and 12 proteins out of the 19 human proteins used as queries, respectively, while G. lamblia seems to have the smallest polyadenylation machinery with only six factors. Remarkably, CPSF30, CPSF73, CstF77, PABP2, and PAP, which were found in all parasites, could represent the core polyadenylation machinery. Multiple genes were detected for several proteins in Entamoeba, while gene redundancy is lower in Giardia and Cryptosporidium. Congruently with their relevance in the polyadenylation process, CPSF73 and PAP are present in all parasites, and CFIm25 is only missing in Giardia. They conserve the functional domains and predicted folding of human proteins, suggesting they may have the same roles in polyadenylation.

CagL polymorphisms D58/K59 are predominant in Helicobacter pylori strains isolated from Mexican patients with chronic gastritis.

BACKGROUND: Helicobacter pylori is a Gram-negative bacterium that colonizes the gastric mucosa in humans. One of the main virulence factors of H. pylori is the cag pathogenicity island (cagPAI), which encodes a type 4-secretion system (T4SS) and the cytotoxin CagA. Translocation of CagA through the T4SS triggers host-signaling pathways. One of the T4SS proteins is CagL, which is necessary for CagA translocation. CagL is a 26-kDa protein that contains a hypervariable motif, which spans residues 58 to 62. Several polymorphisms in this region have been associated with different disease outcomes, e.g. in Mexico, N58 is associated with a higher risk of gastric cancer. The aim of this work is to analyze the sequence of the hypervariable motif (residues 58 to 62) of clinical isolates from Mexican patients with chronic gastritis, and to correlate these polymorphisms with the vacA genotype. RESULTS: Of the 164 biopsies analyzed, only 30.5% (50/164) were positive for H. pylori. Thirty-six of the 50 clinical isolates (72%) were cagA positive, and 40 (80%) had the most virulent vacA genotype (s1/m1). Of the cagA positive strains, 94.4% were vacA s1/m1. All the cagA + strains contained the cagL gene. The most prevalent sequence in the polymorphic region (residues 58-62) was DKMGE (75.8%, 25/33), followed by NKMGQ and NEIGQ (6.1%, 2/33), and DEIGQ, NKMGE, DKIGE, and DKIGK (3%, 1/33). Regarding polymorphisms in positions 58 and 59, the most common were D58/K59 (81.8%, 27/33), followed by N58/K59 (9.1%, 3/33), and D58/E59 (3%, 1/33). Only two isolates (6.1%) contained residues N58/E59, which correspond to those found in H. pylori strain ATCC 26695. 92.6% of the clinical isolates having polymorphism D58/K59 had the genotype vacA s1/m1, considered to be the most virulent, while 7.4% had the genotypes vacA s1/m2 and s2/m2. CONCLUSIONS: In Mexican patients, CagL polymorphisms D58, K59, M60, E62, K122, and I134 are more common in patients with chronic gastritis.

Advances on Aptamers against Protozoan Parasites.

Aptamers are single-stranded DNA or RNA sequences with a unique three-dimensional structure that allows them to recognize a particular target with high affinity. Although their specific recognition activity could make them similar to monoclonal antibodies, their ability to bind to a large range of non-immunogenic targets greatly expands their potential as tools for diagnosis, therapeutic agents, detection of food risks, biosensors, detection of toxins, drug carriers, and nanoparticle markers, among others. One aptamer named Pegaptanib is currently used for treating macular degeneration associated with age, and many other aptamers are in different clinical stages of development of evaluation for various human diseases. In the area of parasitology, research on aptamers has been growing rapidly in the past few years. Here we describe the development of aptamers raised against the main protozoan parasites that affect hundreds of millions of people in underdeveloped and developing countries, remaining a major health concern worldwide, i.e. Trypanosoma spp., Plasmodium spp., Leishmania spp., Entamoeba histolytica, and Cryptosporidium parvuum. The latest progress made in this area confirmed that DNA and RNA aptamers represent attractive alternative molecules in the search for new tools to detect and treat these parasitic infections that affect human health worldwide.

Quercetina atenúa la virulencia de Staphylococcus aureus al disminuir la secreción de alfa toxina / Quercetin attenuates Staphylococcus aureus virulence by reducing alpha-toxin secretion

Alfa toxina, una proteína formadora de poros con actividad citotóxica, es uno de los principales factores de virulencia secretados por la mayoría de las cepas de Staphylococcus aureus. Se ha establecido la relevancia de esta proteína en la patogenia de la neumonía asociada a infecciones por S. aureus. Por lo tanto, la inhibición de la secreción de alfa toxina puede ser una alternativa en el control de las infecciones causadas por este microorganismo. En este trabajo mostramos que quercetina, un flavonoide de origen natural, inhibe de manera dosis dependiente la actividad hemolítica y disminuye la secreción de alfa toxina en sobrenadantes de cultivos de S. aureus sensible y resistente a meticilina. Además, quercetina previene de manera significativa el daño de células alveolares humanas cuando se co-cultivan con S. aureus. Nuestros datos sugieren que quercetina puede disminuir la virulencia de S. aureus al afectar la secreción de alfa toxina.

Alpha toxin, a pore-forming protein with cytotoxic activity, is one of the major virulence factors secreted by most strains of Staphylococcus aureus. The relevance of this protein in the pathogenesis of pneumonia associated with S. aureus infections has already been esta blished. Therefore, inhibiting alpha toxin secretion can be an alternative for controlling these infections. This study shows that quercetin, a naturally occurring flavonoid, inhibits hemolytic activity in a dose-dependent manner and reduces alpha toxin secretion in culture supernatants of methicillin-sensitive and methicillin-resistant S. aureus. Furthermore, quercetin significantly prevents damage to human alveolar cells when co-cultured with S. aureus. Our results suggest that quercetin can reduce S. aureus virulence by affecting alpha-toxin secretion.

[Quercetin attenuates Staphylococcus aureus virulence by reducing alpha-toxin secretion]. / Quercetina atenúa la virulencia de Staphylococcus aureus al disminuir la secreción de alfa toxina.

Alpha toxin, a pore-forming protein with cytotoxic activity, is one of the major virulence factors secreted by most strains of Staphylococcus aureus. The relevance of this protein in the pathogenesis of pneumonia associated with S.aureus infections has already been established. Therefore, inhibiting alpha toxin secretion can be an alternative for controlling these infections. This study shows that quercetin, a naturally occurring flavonoid, inhibits hemolytic activity in a dose-dependent manner and reduces alpha toxin secretion in culture supernatants of methicillin-sensitive and methicillin-resistant S.aureus. Furthermore, quercetin significantly prevents damage to human alveolar cells when co-cultured with S.aureus. Our results suggest that quercetin can reduce S.aureus virulence by affecting alpha-toxin secretion.

Silencing the cleavage factor CFIm25 as a new strategy to control Entamoeba histolytica parasite.

The 25 kDa subunit of the Clevage Factor Im (CFIm25) is an essential factor for messenger RNA polyadenylation in human cells. Therefore, here we investigated whether the homologous protein of Entamoeba histolytica, the protozoan responsible for human amoebiasis, might be considered as a biochemical target for parasite control. Trophozoites were cultured with bacterial double-stranded RNA molecules targeting the EhCFIm25 gene, and inhibition of mRNA and protein expression was confirmed by RT-PCR and Western blot assays, respectively. EhCFIm25 silencing was associated with a significant acceleration of cell proliferation and cell death. Moreover, trophozoites appeared as larger and multinucleated cells. These morphological changes were accompanied by a reduced mobility, and erythrophagocytosis was significantly diminished. Lastly, the knockdown of EhCFIm25 affected the poly(A) site selection in two reporter genes and revealed that EhCFIm25 stimulates the utilization of downstream poly(A) sites in E. histolytica mRNA. Overall, our data confirm that targeting the polyadenylation process represents an interesting strategy for controlling parasites, including E. histolytica. To our best knowledge, the present study is the first to have revealed the relevance of the cleavage factor CFIm25 as a biochemical target in parasites.

Aptamers as a promising approach for the control of parasitic diseases

ABSTRACT Aptamers are short single-stranded RNA or DNA oligonucleotides that are capable of binding various biological targets with high affinity and specificity. Their identification initially relies on a molecular process named SELEX (Systematic Evolution of Ligands by EXponential enrichment) that has been later modified in order to improve aptamer sensitivity, minimize duration and cost of the assay, as well as increase target types. Several biochemical modifications can help to enhance aptamer stability without affecting significantly target interaction. As a result, aptamers have generated a large interest as promising tools to compete with monoclonal antibodies for detection and inhibition of specific markers of human diseases. One aptamer-based drug is currently authorized and several others are being clinically evaluated. Despite advances in the knowledge of parasite biology and host-parasite interactions from "omics" data, protozoan parasites still affect millions of people around the world and there is an urgent need for drug target discovery and novel therapeutic concepts. In this context, aptamers represent promising tools for pathogen identification and control. Recent studies have reported the identification of "aptasensors" for parasite diagnosis, and "intramers" targeting intracellular proteins. Here we discuss various strategies that have been employed for intracellular expression of aptamers and expansion of their possible application, and propose that they may be suitable for the clinical use of aptamers in parasitic infections.

Aptamers as a promising approach for the control of parasitic diseases.

Aptamers are short single-stranded RNA or DNA oligonucleotides that are capable of binding various biological targets with high affinity and specificity. Their identification initially relies on a molecular process named SELEX (Systematic Evolution of Ligands by EXponential enrichment) that has been later modified in order to improve aptamer sensitivity, minimize duration and cost of the assay, as well as increase target types. Several biochemical modifications can help to enhance aptamer stability without affecting significantly target interaction. As a result, aptamers have generated a large interest as promising tools to compete with monoclonal antibodies for detection and inhibition of specific markers of human diseases. One aptamer-based drug is currently authorized and several others are being clinically evaluated. Despite advances in the knowledge of parasite biology and host-parasite interactions from "omics" data, protozoan parasites still affect millions of people around the world and there is an urgent need for drug target discovery and novel therapeutic concepts. In this context, aptamers represent promising tools for pathogen identification and control. Recent studies have reported the identification of "aptasensors" for parasite diagnosis, and "intramers" targeting intracellular proteins. Here we discuss various strategies that have been employed for intracellular expression of aptamers and expansion of their possible application, and propose that they may be suitable for the clinical use of aptamers in parasitic infections.

Amino acid residues Leu135 and Tyr236 are required for RNA binding activity of CFIm25 in Entamoeba histolytica.

Pre-mRNA 3' end processing in the nucleus is essential for mRNA stability, efficient nuclear transport, and translation in eukaryotic cells. In Human, the cleavage/polyadenylation machinery contains the 25 kDa subunit of the Cleavage Factor Im (CFIm25), which specifically recognizes two UGUA elements and regulates the assembly of polyadenylation factors, poly(A) site selection and polyadenylation. In Entamoeba histolytica, the protozoan parasite responsible for human amoebiasis, EhCFIm25 has been reported as a RNA binding protein that interacts with the Poly(A) Polymerase. Here, we follow-up with the study of EhCFIm25 to characterize its interaction with RNA. Using in silico strategy, we identified Leu135 and Tyr236 in EhCFIm25 as conserved amino acids among CFIm25 homologues. We therefore generated mutant EhCFIm25 proteins to investigate the role of these residues for RNA interaction. Results showed that RNA binding activity was totally abrogated when Leu135 and Tyr236 were replaced with Ala residue, and Tyr236 was changed for Phe. In contrast, RNA binding activity was less affected when Leu135 was substituted by Thr. Our data revealed for the first time -until we know-the functional relevance of the conserved Leu135 and Tyr236 in EhCFIm25 for RNA binding activity. They also gave some insights about the possible chemical groups that could be interacting with the RNA molecule.